JP6936987B2 - Target substance detection chip, target substance detection device and target substance detection method - Google Patents

Description

The present invention relates to a target substance detection chip, a target substance detection device, and a target substance detection method capable of holding a plurality of liquid samples at the same time and observing them independently in optical observation using surface plasmon resonance and waveguide mode.

Recently, health diagnostic, pharmaceutical, early detection of diseases and infectious diseases, environmental pollution detection, in various fields, such as counter-terrorism, can carry, operation is required simple and sensitive detector. SPR sensors that use surface plasmon resonance (SPR) and waveguide mode sensors that use waveguide mode are known as sensors that are small enough to be carried and can measure various substances contained in liquids. (See, for example, Non-Patent Documents 1 to 19 and Patent Documents 1 to 7). These sensors detect various biomarkers and viruses caused by diseases, selectively detect various biomaterials such as proteins, evaluate pollution in the environment, and detect toxic substances, illegal drugs, and explosives used in terrorism. Has been used in.

As the SPR sensor, a Klechman-arranged SPR sensor is widely used.
In the Klechman-arranged SPR sensor, a metal such as gold, silver, or aluminum is vapor-deposited on a transparent substrate to form a metal thin film layer, and optical is applied to a surface of the transparent substrate opposite to the surface on which the metal thin film layer is formed. It has a structure in which prisms are in close contact with each other, and the laser beam emitted from the light source is polarized by a polarizing plate and irradiated to the transparent substrate via the optical prism. The incident light is incident under the condition of total internal reflection. Surface plasmon resonance is expressed at a specific angle of incidence by the evanescent wave that exudes from the incident light to the surface side of the metal thin film layer. When the surface plasmon resonance occurs, the evanescent wave is absorbed by the surface plasmon, so that the intensity of the reflected light is remarkably reduced in the vicinity of this incident angle. Since the conditions under which the surface plasmon resonance occurs change depending on the dielectric constant near the surface of the metal thin film layer, when the surface of the metal thin film layer is adsorbed, approached, detached, or altered, the intensity of reflected light is increased. Changes appear. Therefore, when the target substance is bonded or adsorbed on the surface of the metal thin film layer and the dielectric constant changes, the reflection characteristics of the incident light change, so that the reflected light reflected from the metal thin film layer changes. The target substance can be detected by monitoring the change in intensity with an optical detector.

The waveguide mode sensor has a structure similar to that of the SPR sensor, and is a sensor that detects the adsorption of substances and changes in the dielectric constant on the detection surface. It is known that this waveguide mode sensor can use an optical system equivalent to all the optical systems that can be used in the SPR sensor.
In a waveguide mode sensor using an arrangement similar to the Kletchman arrangement, a transparent substrate, a thin film layer composed of a metal layer or a semiconductor layer coated on the transparent substrate, and a dielectric layer formed on the thin film layer are used. A detection plate consisting of is used. The optical prism is brought into close contact with the surface of the detection plate opposite to the surface on which the dielectric layer is formed via the refractive index adjusting oil. The incident light emitted from the light source and polarized by the polarizing plate is applied to the detection plate via the optical prism. The incident light is incident on the detection plate under the condition of total internal reflection. When the incident light is combined with a waveguide mode (also called leakage mode or leaky mode) propagating in the dielectric layer at a specific incident angle, the waveguide mode is excited and the light is emitted in the vicinity of this incident angle. The reflected light intensity changes greatly. Since the excitation conditions of such a waveguide mode change depending on the dielectric constant near the surface of the dielectric layer, when the target substance is adsorbed, approached, separated, or altered on the surface of the dielectric layer, the reflected light is reflected. A change appears in the intensity of. By observing this change with a photodetector, it is possible to detect phenomena such as adsorption, approach, detachment, and alteration of the target substance.

Further, when a fluorescent substance (for example, a fluorescent dye) capable of photoexcited light emission is attached to or brought close to the detection surface of the SPR sensor or the waveguide mode sensor, there is also an effect of enhancing the light emission of the fluorescent substance. That is, when one surface of the detection plate is irradiated with light under total reflection conditions, an enhanced electric field is formed on the other surface, and the fluorescent substance can be strongly emitted by using the enhanced electric field as excitation light. Fluorescence observation with less background light can be performed (see, for example, Patent Document 8).
When a light scattering substance (for example, nanoparticles composed of a scattering body such as polystyrene beads or gold nanoparticles) is attached to or brought close to the detection surface of the SPR sensor or the waveguide mode sensor, the light scattering substance is subjected to. It also has the effect of enhancing scattered light. That is, when one surface of the detection plate is irradiated with light under total reflection conditions, an enhanced electric field is formed on the other surface, and the enhanced electric field can generate strong scattered light from the light scattering substance. , It is possible to observe scattered light with high brightness (see, for example, Non-Patent Document 20).
Various methods have been proposed as a method of generating an electric field enhancement by total internal reflection of light to obtain an enhanced electric field, and the present inventor has a detection plate in which a _{silicon layer and a SiO 2 layer are laminated in this order on a silica glass substrate.} Is proposed on a trapezoidal prism made of silica glass, and light is irradiated through the prism under the total reflection condition on the surface of the detection plate to obtain an enhanced electric field (see Non-Patent Document 21).

By the way, the present inventor holds a plurality of liquid samples at the same time and observes them independently for the purpose of reducing the operational burden when using the SPR sensor and the waveguide mode sensor and performing efficient observation. We have proposed a multi-channel type detection plate capable of performing the above (see Patent Document 9).
However, in the proposal of this multi-channel type detection plate, since the liquid sample is introduced near the surface of the detection chip, a concave accommodating portion for accommodating the detection chip or a flow for sending the liquid sample to the detection chip. It is necessary to prepare a structure that is not directly involved in the formation of the enhanced electric field such as a path.
Therefore, at present, there is a demand for the development of a new detection chip that can be manufactured in a smaller size and at a lower cost by reducing the number of parts.

Japanese Patent No. 4581135 Japanese Patent No. 4595072 Japanese Unexamined Patent Publication No. 2007-271596 Japanese Unexamined Patent Publication No. 2008-46093 Japanese Unexamined Patent Publication No. 2009-85714 International Publication No. 2010/87088 JP-A-2010-145408 International Publication 2015/194663 Japanese Patent No. 5923811

W.Knoll, MRS Bulletin 16, pp.29-39 (1991) W.Knoll, Annu.Rev.Phys.Chem.49, pp.569-638 (1998) H.Kano et al., Appl.Opt.33, pp.5166-5170 (1994) C. Nylander et al. Sensor.Actuat.3, pp.79-88 (1982/83) K.Kambhampatiet al. Langmuir 17, pp.1169-1175 (2001) O.R.Bolduc et al. Talanta 77, pp.1680-1687 (2009) I. Stammler et al. Sensor.Actuat.B54, pp98-105 (1999) M.Osterfeld et al. Appl.Phys.Lett.62, pp.2310-2312 (1993) E.F.Aust et al. J.Appl.Phys.73, p.2705 (1993) M. Fujimaki et al. Microelectronic Engineering 84, pp.1685-1689 (2007) K.Awazu et al. Optics Express 15, pp.2592-2597 (2007) K.H.A.Lau et al. J.Phys.Chem.B108, pp.10812 (2004) M.Fujimaki et al. Optics Express 16, pp.6408-6416 (2008) M.Fujimaki et al. Nanotechnology 19, pp.095503-1-095503-7 (2008) M.Fujimaki et al. J.Microscopy 229, pp.320-326 (2008) M.Fujimaki et al. Optics Express 18, pp.15732-15740 (2010) R.P.Podgorsek et al. Sensor.Actuat.B51 pp.146-151 (1998) J.J.Saarinen et al. Opt.Express 13, pp.3754-3764 (2005) G.Rong et al. Biosens.Bioelectron.23, pp.1572-1576 (2008) M.Yasuura and M.Fujimaki, Sci. Rep. Vol. 6, pp. 39241-1-39241-7 (2016) M.Fujimaki et al. Optics Express, Vol. 23 (2015) pp.10925-10937

The present invention solves the above-mentioned problems in the prior art, and provides a multi-channel type target substance detection chip that can be manufactured in a small size and at low cost, a target substance detection device using the same, and a target substance detection method. Make it an issue.

The means for solving the above-mentioned problems are as follows. That is,
<1> A light-transmitting substrate and a light-transmitting substrate formed on the light-transmitting substrate, and either surface plasmon resonance or waveguide mode can be excited when light is irradiated from one surface under total reflection conditions. When a plurality of electric field enhancing layers are formed on any surface of the light transmissive substrate, the surface on which the electric field enhancing layer is formed, and the other surface of the electric field enhancing layer, and viewed from the other surface. A closed water-repellent portion having a shape drawn by a closed figure surrounding the electric field enhancing layer is arranged, and the closed figure is drawn in a regular hexagonal shape by a linear film having a width of 5 μm to 20 μm, and the adjacent closed figure is drawn. A target substance detection chip characterized in that water portions are formed so as to share one side of the regular hexagon .
< 2 > A plurality of electric field enhancing layers are formed scattered on the light transmissive substrate, and closed water repellent portions are formed on the surface of the light transmissive substrate on which the electric field enhancing layer is formed. The target substance detection chip according to <1>, wherein one electric field enhancing layer is formed in the closed figure of the closed water repellent portion so as to be separated from the drawn line of the closed figure.
< 3 > The target substance detection chip according to any one of <1> to <2 >, wherein the maximum diameter of the closed figure is at most 5 mm.
< 4 > The target substance detection chip according to any one of <1> to < 2 > and the surface of the target substance detection chip on which the closed water-repellent portion is formed are used as the front surface, and the electric field enhancing layer is formed from the back surface side. A light irradiation unit capable of irradiating one surface with light under total reflection conditions, and a reflection arranged on the back surface side of the target substance detection chip and capable of detecting reflected light reflected from the electric field enhancing layer. A target substance detection device characterized in that a light detection unit and a light detection unit are arranged.
< 5 > The target substance detection chip according to any one of <1> to < 3 > and the surface of the target substance detection chip on which the closed water-repellent portion is formed are used as the front surface, and the electric field enhancing layer is formed from the back surface side. A light irradiation unit capable of irradiating one surface with light under all reflection conditions, and a liquid sample arranged on the surface side of the target substance detection chip and held in the closed water repellent portion based on the light irradiation. A target substance detection device, characterized in that a light detection unit capable of detecting fluorescence or scattered light emitted from a target substance contained in the target substance or a labeling substance bound to the target substance is arranged.
< 6 > A target substance detection method for detecting a target substance using the target substance detection chip according to any one of <1> to < 3 >, wherein a liquid sample is placed in a closed water-repellent portion of the target substance detection chip. The liquid sample introduction step to be introduced, the light irradiation step of irradiating one surface of the electric field enhancing layer from the back surface side of the target substance detection chip with light under all reflection conditions, and the light reflected from the electric field enhancing layer are detected. A method for detecting a target substance, which comprises a step of detecting reflected light.
< 7 > A target substance detection method for detecting a target substance using the target substance detection chip according to any one of <1> to < 3 >, wherein a liquid sample is placed in a closed water-repellent portion of the target substance detection chip. It is included in the liquid sample based on the liquid sample introduction step to be introduced, the light irradiation step of irradiating one surface of the electric field enhancing layer from the back surface side of the target substance detection chip with light under the total reflection condition, and the light irradiation. A target substance detection method comprising a light detection step of detecting fluorescence or scattered light emitted from a target substance or a labeling substance bound to the target substance.

According to the present invention, the above-mentioned problems in the prior art can be solved, and a multi-channel type target substance detection chip that can be manufactured in a small size and at low cost, a target substance detection device using the same, and a target substance detection method. Can be provided.

It is sectional drawing which shows the schematic structure of the target substance detection chip which concerns on one Embodiment of this invention. It is explanatory drawing which shows the state when a part of the target substance detection chip shown in FIG. 1 is magnified and viewed from the other surface of the electric field strengthening layer. It is a cross-sectional view which shows the structure corresponding to one closed water-repellent part 4 enlarged. It is explanatory drawing which shows the schematic structure of 1st Embodiment. It is explanatory drawing which shows the schematic structure of 2nd Embodiment. It is explanatory drawing which shows the schematic structure of 3rd Embodiment.

(Target substance detection chip)
The target substance detection chip of the present invention is configured by arranging a light-transmitting substrate, an electric field enhancing layer, and a closed water-repellent portion.

<Light transmissive substrate>
The light-transmitting substrate is a member that supports the electric field-enhancing layer and transmits light irradiated to the electric field-enhancing layer.
The light transmissive substrate is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a known light transmissive substrate such as a glass substrate or a plastic substrate can be used.
In addition, in this specification, "light transmittance" means that the visible light transmittance is 0.5% or more.

<Electric field enhancement layer>
The electric field enhancing layer is a layer in which either surface plasmon resonance or waveguide mode can be excited when light is irradiated from one surface under total reflection conditions.
The electric field enhancing layer is not particularly limited and may be appropriately selected depending on the intended purpose, and known surface plasmon excitation layers and waveguide mode excitation layers can be applied.

Examples of the surface plasmon excitation layer include a metal layer 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 irradiating the one surface, and the enhanced electric field is obtained on the other surface.
The optimum value of the thickness of the metal layer is determined by the constituent material and the wavelength of the irradiating light, and it is known that this value can be calculated by calculation using Fresnel's equation. Generally, 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 of nm.

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

When observing light emission from a target substance or a fluorescent substance that labels the target substance, when the target substance and the fluorescent substance are close to the metal layer, the energy obtained by the target substance or the fluorescent substance from the excitation light is generated. A phenomenon called quenching may occur in which the light is transferred to the metal layer and the luminous efficiency is lowered.
In this case, when a coating layer is formed on the surface of the metal layer for the purpose of separating the target substance and the fluorescent substance from the surface of the metal layer, the quenching is suppressed and the decrease in luminous efficiency is suppressed. Can be done.
The coating layer is not particularly limited, and can be formed of a transparent layer having a thickness of several nm to several tens of nm formed of a glass material such as silica glass or an organic polymer material.

The waveguide mode excitation layer is not particularly limited, and examples thereof include a laminate of a thin film layer formed of a metal material or a semiconductor material and a dielectric layer formed of a light-transmitting dielectric material.
In the waveguide mode excitation layer, the waveguide mode is excited in the dielectric layer by the light irradiating the one surface, and the enhanced electric field is obtained on the other surface.
In the waveguide mode excitation layer, the thin film layer constitutes the one surface side layer, and the dielectric layer constitutes the other surface side.

The metal material is not particularly limited, and examples thereof include gold, silver, copper, platinum, and aluminum.
The semiconductor material is not particularly limited, and examples thereof include semiconductor materials such as silicon and germanium, and known compound semiconductor materials. Among them, silicon, which is inexpensive and easy to process, is preferable.
The thickness of the thin film layer is the same as that of the surface plasmon excitation layer, and an optimum value is determined by the constituent material and the wavelength of the irradiated light, and this value can be calculated from the calculation using Fresnel's equation. It is known. Generally, when light in the wavelength band from near-ultraviolet to near-infrared is used, the thickness of the thin film layer is several nm to several hundred 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. Silicon oxide is preferable because it is easy to use and has high chemical stability. In this case, if the thin film layer is formed of the silicon, it can be easily formed by oxidizing the surface side of the silicon layer.
The thin film layer and the dielectric layer can be appropriately selected from known forming methods depending on the material.

<Closed water repellent part>
A plurality of the closed water-repellent portions are formed on any surface of the light-transmitting substrate on which the electric field enhancing layer is formed and the other surface of the electric field enhancing layer, and are viewed from above the other surface. At that time, the shape is drawn by a closed figure surrounding the electric field enhancing layer.

As described above, the closed water-repellent portion may be formed on the surface of the light-transmitting substrate on which the electric field enhancing layer is formed, or may be formed on the other surface of the electric field enhancing layer. ..
In the former case, the closed water-repellent portion is formed on the light-transmitting substrate, and the electric field enhancing layer is formed in the closed figure of the closed water-repellent portion. The order of formation is not particularly limited, and the electric field enhancing layer may be formed after the closed water repellent portion is formed, or the closed water repellent portion may be formed after the electric field enhancing layer is formed. ..
Further, in the latter case, it can be configured by forming the electric field enhancing layer on the light transmissive substrate and then forming the closed water repellent portion on the electric field enhancing layer.

The method for forming the closed water-repellent portion is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a linear film of a known fluorine-based, silicone-based, or acrylic-based water-repellent compound is vapor-deposited. It may be formed by a known forming method such as a method, a sputtering method, a coating method, or a printing method. Further, a known fluorine coating method of irradiating a known fluorine-based water-repellent gas with plasma can be mentioned.

As an example, when the closed water-repellent portion is formed by the printing method, there is no particular limitation, but there is a known water repellency containing a silicone-based surfactant or a fluorine-based surfactant together with a binder resin, a solvent, a reactive diluent and the like. Ink can be used.
The content of each of the surfactants in the water-repellent ink is not particularly limited and is about 0.5% by mass to 20% by mass.

The silicone-based surfactant is not particularly limited and may be appropriately selected from known ones. For example, a silicone oil having a dimethylsiloxane skeleton, a silicone resin, and a part of these methyl groups are alkyl. Examples thereof include modified silicone oils and modified silicone resins substituted with groups, aryl groups, alkoxy groups, hydroxyl groups and the like.
The fluorosurfactant is also not particularly limited and may be appropriately selected from known ones. For example, a monomer having a perfluoroalkyl group is reacted with a monomer having various reactive groups. Examples thereof include polymers and oligomers having a perfluoroalkyl group in the side chain.

The binder resin is not particularly limited, and can be arbitrarily selected from polymer compounds that are compatible with the silicone-based surfactant or the fluorine-based surfactant and become solid after drying.
Examples of the polymer compound include acrylic resins, epoxy resins, melamine resins, urethane resins, polyurethane resins, polyester resins, butyral resins, polyvinyl alcohol resins, acetal resins, phenol resins, nitrocellulose, ethyl cellulose and other cellulose-based resins. Examples thereof include chlorinated rubber, petroleum resin, vinylidene fluoride resin and the like.

Any printing method can be adopted as the drawing (patterning) method using the water-repellent ink, but when the pattern accuracy is about 5 μm to 20 μm width, the letterpress inversion printing method is preferable, and the pattern accuracy is high. When it exceeds 20 μm, a gravure printing method, a gravure offset printing method, a flexo printing method, an offset printing method, a screen printing method and the like are preferable.
For example, when the viscosity of the water-repellent ink is adjusted to about 1 Pa · s to 20 Pa · s and the gravure printing method is adopted, the pattern accuracy is about 20 μm ± 5 μm and the film thickness is about 1.5 μm to 2.5 μm. A patterned layer can be obtained.

The closed figure is a plane figure having no open end in which the start end and the end of the line are connected without overlapping, and can be selected from any plane figure such as a polygonal shape, a circular shape, and an elliptical shape. Can be done. Among these, the polygonal shape is preferable.
When the closed figure has the polygonal shape, the adjacent closed water-repellent portions are formed so as to share one side of the polygonal shape, and many closed water-repellent portions can be formed in a limited area. It is possible to reduce the size while securing the required number of channels.
Further, if the polygonal shape is any one of an equilateral triangle, an isosceles triangle, a right triangle, a rhombus, a parallelogram, a rectangle, a regular quadrangle, a regular hexagon, and a regular octagon, a plurality of the closed water repellency in a limited area. The portions can be arranged closely side by side, and even more closed water-repellent portions can be formed.
The polygonal shape means a shape in which the largest internal angle is less than 180 ° and is drawn by connecting straight lines having a length of at least 1 μm.

The size of the closed water-repellent portion is not particularly limited and depends on the spot diameter of the light irradiating the one surface of the electric field enhancing layer, etc. A closed water-repellent portion can be formed, and the maximum diameter of the closed figure is preferably 5 mm at the maximum.

Hereinafter, an example of the embodiment of the target substance detection chip will be described with reference to the drawings.
FIG. 1 is a cross-sectional view showing a schematic configuration of the target substance detection chip according to an embodiment of the present invention. Further, FIG. 2 is an explanatory view showing a state when a part of the target substance detection chip shown in FIG. 1 is magnified and viewed from above the other surface of the electric field enhancing layer 3.

As shown in FIGS. 1 and 2, the target substance detection chip 1 is configured by arranging a light transmissive substrate 2, a plurality of electric field enhancing layers 3, and a plurality of closed water repellent portions 4.
Further, in the target substance detection chip 1, a plurality of electric field enhancing layers 3 are formed scattered on the light transmissive substrate 2, and the closed water repellent portion 4 is formed by the electric field enhancing layer 3 of the light transmissive substrate 2. One electric field enhancing layer 3 is formed in the closed figure of the closed water repellent portion 4 on the surface to be formed, apart from the drawn line of the closed figure.

Further, as the closed water repellent portion 4, the closed figure has a regular hexagonal shape, and two closed water repellent portions 4 adjacent to each other on all sides constituting the regular hexagon share one side of the regular hexagon. It is formed.
Therefore, in the target substance detection chip 1, many closed water repellent portions 4 are formed by closely arranging a plurality of closed water repellent portions 4 in a limited area.

The maximum diameter L ₁ in the closed figure of the closure repellent portion 4, the smaller, it is possible to form a plurality of closed water-repellent portion 4 in a limited area, is preferably 5mm even larger.
Further, the electric field enhancing layer 3 is arranged at the center position of the closed figure so that one electric field enhancing layer 3 is formed in the closed figure of the closed water repellent portion 4 so as to be separated from the drawn line of the closed figure. It is preferable that the maximum diameter L ₂ is less than _{L 1.}

FIG. 3 shows a state in which a liquid sample is introduced into the target substance detection chip 1. Note that FIG. 3 is an enlarged cross-sectional view showing a structure corresponding to one closed water repellent portion 4.
When the liquid sample 5 is dropped and introduced into the closed water-repellent portion 4 of the target substance detection chip 1, the liquid sample 5 is introduced into the closed water-repellent portion 4 due to the water repellency of the closed water-repellent portion 4, as shown in FIG. Be retained.
Therefore, the target substance detection chip 1 is a multi-channel type detection chip in which different types of liquid samples 5 are simultaneously held by the plurality of closed water repellent portions 4, and each liquid sample 5 can be observed independently.

In the target substance detection chip 1 configured in this way, it is not necessary to fabricate a structure that is not directly involved in the formation of the augmented electric field such as the concave accommodating portion and the flow path in the prior art, and the number of parts can be reduced. It is small and can be manufactured at low cost.

(Target substance detector)
The target substance detection chip of the present invention includes a target substance detection device that detects reflected light from the electric field enhancing layer and the target substance or the target substance contained in the liquid sample held on the target substance detection chip. It can be applied to any of the target substance detection devices that detect fluorescence or scattered light from the bound labeling substance.
The first target substance detection device of the present invention is configured by arranging the target substance detection chip of the present invention, a light irradiation unit, and a reflected light detection unit in the former aspect.
The second target substance detection device of the present invention is configured by arranging the target substance detection chip of the present invention, a light irradiation unit, and a light detection unit in the latter aspect.
First, the first target substance detection device of the present invention will be described.

<Light irradiation part>
The light irradiation unit can irradiate one surface of the electric field enhancing layer from the back surface side with the surface of the target substance detection chip on which the closed water repellent portion is formed as the front surface under total reflection conditions. Will be done.
The light source of the light irradiation unit is not particularly limited and may be appropriately selected depending on the intended purpose, and examples thereof include known lamps, LEDs, and lasers.

When a radiation light source such as a lamp or LED is used, irradiation of the irradiation light is performed so that the light in all directions of the emitted light radiating to the back surface side of the target substance detection chip satisfies the total reflection condition. A guide unit such as a collimating lens that regulates the direction to a specific direction may be used.

Here, when the front surface and the back surface of the target substance detection chip are parallel plates, the light emitted from the back surface side is not totally reflected when the liquid is present on the front surface. Therefore, in such a case, by forming a diffraction grating on the back surface portion of the target substance detection chip, when the diffraction grating is irradiated with light at a specific angle, the light is diffracted by the diffraction grating. The object is introduced into the target substance detection chip, and the light introduced into the target substance detection chip is irradiated to the surface under all reflection conditions to form the enhanced electric field on the surface. A substance detection chip may be configured. Alternatively, the front surface and the back surface may be formed so as not to be parallel to each other. Alternatively, the light emitted from the light source may be applied to the back surface of the target substance detection chip via a known prism. The prism can be used by being optically bonded to the back surface of the target substance detection chip with a refractive index adjusting oil, an optical adhesive, or the like. Further, when the same forming material as the forming material of the light transmitting substrate is selected as the forming material of the prism, a material in which the light transmitting substrate and the prism are integrally molded can also be used.

<Reflected light detector>
The reflected light detection unit is arranged on the back surface side of the target substance detection chip, and can detect the reflected light reflected from the electric field enhancing layer.
The reflected light detection unit is not particularly limited, and can be configured by using a known photodetector such as a thermopile sensor, a diode sensor, a CMOS sensor, or a CCD sensor, and an image sensor such as a CMOS sensor or a CCD sensor can be used. When used, a plurality of closed water-repellent portions 4 can be observed at the same time, which is preferable.

In the first target substance detection device, when the electric field enhancing layer is formed by the surface plasmon excitation layer, when the one surface of the electric field enhancing layer is irradiated with light under total reflection conditions, the light transmissive substrate Evanescent light exudes from the electric field-enhancing layer toward the electric field-enhancing layer, and the surface plasmon is excited in the vicinity of the surface of the target substance detection device.
Here, when the light incident angle from the light irradiation unit is adjusted, surface plasmon resonance in which the evanescent light is absorbed by the surface plasmon occurs at a specific incident angle θ, and the intensity of the reflected light from the electric field enhancing layer is increased. Significantly reduced. The reflected light intensity is detected for each incident angle by the reflected light detecting unit.
Since the incident angle θ changes in shift according to the change in the dielectric constant in the vicinity of the surface, it is possible to detect the target substance in the liquid sample held on the surface through the shift change in the incident angle θ. can.
Further, when the reflected light intensity is observed by the reflected light detection unit in a state where the incident angle θ is fixed near the angle at which surface plasmon resonance appears, the reflected light intensity corresponds to the change in the dielectric constant in the vicinity of the surface. Changes occur, and changes in light and darkness are observed. Therefore, the target substance in the liquid sample held on the surface can be detected through this change in brightness.

In the first target substance detection device, when the electric field enhancing layer is formed by the waveguide mode excitation layer, when light is incident at a specific incident angle θ while satisfying the total reflection conditions, the thin film layer. And the waveguide mode in which the propagating light propagates in the dielectric layer is excited. In the vicinity of this incident angle θ, the reflected light intensity is significantly different from that of other incident angles. Further, the excitation conditions of the waveguide mode are sensitive to a change in the dielectric constant on the surface of the target substance detection chip, and when the dielectric constant changes due to adsorption of the target substance or the like, the above-mentioned near the incident angle θ. It appears as a fluctuation in the reflection characteristics of the reflected light.
At this time, the adsorption of the target substance and the like are held on the surface by the reflected light detection unit capturing a sudden decrease in the reflected light intensity at a specific incident angle θ when the monochromatic light is incident. The target substance in a liquid sample can be detected.
Further, when the reflected light intensity is observed by the reflected light detection unit in a state where the incident angle θ is fixed near the angle at which the waveguide mode is excited, the reflected light is observed according to the change in the dielectric constant in the vicinity of the surface. There is a change in intensity, and a change in light and darkness is observed. Therefore, the target substance in the liquid sample held on the surface can be detected through this change in brightness.

Next, the second target substance detection device will be described. The light irradiation unit can be appropriately selected from the items described for the first target substance detection device.

<Light detector>
The light detection unit is arranged on the surface side of the target substance detection chip, and is held in the closed water-repellent portion based on the irradiation of the light. The target substance contained in the liquid sample or a label bonded to the target substance. It is possible to detect fluorescence or scattered light emitted from a substance.
The photodetector is not particularly limited and may be appropriately selected depending on the intended purpose, and a known photodetector such as a photodiode or a photomultiplier tube can be used.
If the optical signal information can be acquired as two-dimensional image information, a plurality of closed water-repellent portions 4 can be observed at the same time, and the position information of the optical signal in the two-dimensional image information appearing as a light spot and 2 It is possible to observe the size information observed on the dimension and the increase / decrease information of the optical signal intensity at the light spot in time series. In order to enable such acquisition of two-dimensional image information, an imaging device may be selected as the photodetector.
The image pickup device is not particularly limited and may be appropriately selected depending on the intended purpose, and a known image sensor such as a CCD image sensor or a CMOS image sensor can be used.

The labeling substance is used to label the target substance when the target substance is a substance that does not easily generate fluorescence or scattered light.
The labeling substance is not particularly limited, and examples thereof include a fluorescent labeling substance and a light scattering substance that specifically adsorb or bind to the target substance to label the target substance.
As the fluorescent labeling substance, for example, a known fluorescent substance such as a fluorescent dye, quantum dots, or a fluorescent dye can be used.
Further, as the light scattering substance, for example, known light scattering substances such as nanoparticles, for example, polystyrene beads and gold nanoparticles can be used.
The binding method between the target substance and the labeling substance is not particularly limited, and known binding methods such as physical adsorption, antigen-antibody reaction, DNA hybridization, biotin-avidin binding, chelate binding, and amino binding can be used. Can be applied.

In the second target substance detection device, the second target substance detection device is formed in the vicinity of the other surface (the surface of the target substance detection chip) of the electric field enhancing layer based on the excitation of either the surface plasmon resonance or the waveguide mode. Using the enhanced electric field as excitation light, the target substance or a fluorescent substance that labels the target substance is emitted, or scattered light is generated from the target substance or a light scattering substance that labels the target substance, and the light is generated. The signal is detected by the light detection unit.

In the second target substance detection device, the light detection unit observes the liquid sample from the surface side of the target substance detection chip. At this time, as shown in FIG. 3, the liquid sample exhibits an upward convex shape due to surface tension, so that the lens effect works, and when the image pickup device is used for the light detection unit, the target substance detection chip Focus shift occurs during surface observation, making it difficult to acquire an image. Therefore, it is preferable to arrange a cover glass on the liquid sample to flatten the liquid surface.

By the way, in the second target substance detection device, in order to use the enhanced electric field formed in the vicinity of the surface of the target substance detection chip for detection, the liquid sample is introduced into the closed water repellent portion and then the liquid sample is introduced. It is necessary to wait for the target substance floating in the target substance to gravitationally settle in the vicinity of the surface of the target substance detection chip.
Therefore, when measuring in a short time, magnetic particles that bind to the target substance are added to the liquid sample to form a conjugate, and the conjugate suspended in the liquid sample is used as the target substance detection chip. It is effective to apply a magnetic field that attracts the surface.
Therefore, as the second target substance detection device, it is preferable that a magnetic field application unit capable of applying such a magnetic field is further arranged. Specifically, it is preferable that a magnetic field application portion capable of applying a magnetic field that attracts the conjugate to the front surface of the target substance detection chip is arranged on the back surface side of the target substance detection chip.

Further, in the second target substance detection device, in order to detect an optical signal in the vicinity of the surface of the target substance detection chip, the optical signal includes scattered light due to dirt or scratches on the surface of the target substance detection chip, and the target substance. If noise signals based on autofluorescence generated from the constituent members of the detection chip, light emission from impurities contained in the liquid sample, and the like are included, the detection accuracy is lowered.
Therefore, by using the light detection unit to compare and observe the state of the conjugate in which the magnetic particles are bound to the target substance before and after the application of the magnetic field by the magnetic field application unit, the optical signal before the application of the magnetic field can be obtained. It is effective to perform observation excluding the included noise signal.
That is, while the conjugate moves due to the application of the magnetic field, the noise signal caused by scratches on the surface of the target substance detection chip does not move due to the application of the magnetic field. The noise signal can be eliminated by performing detection focusing on the moving optical signal.
Therefore, as the second target substance detection device, it is preferable that a magnetic field application unit capable of applying such a magnetic field is further arranged. Specifically, it is possible to apply a first magnetic field that moves the conjugate contained in the liquid sample held on the surface of the target substance detection chip in a direction parallel to the surface or away from the surface. It is possible to apply a second magnetic field that is arranged on the back surface side of the first magnetic field application portion and the target substance detection chip and that attracts the conjugate in the liquid sample held on the surface onto the surface. At the same time, the magnetic field is applied to any of the second magnetic field application portions that can move the magnetic particles in a direction having a vector component in a direction parallel to the in-plane direction of the surface while the second magnetic field is applied. It is preferable that the parts are arranged. Above all, it is particularly preferable to arrange the second magnetic field application unit because the attractive magnetic field can be applied for the purpose of measurement in a short time.

The constituent members of the magnetic field application portion are not particularly limited, and known permanent magnets, electromagnets, and the like can be mentioned. Further, as the second magnetic field application unit, a region (detection) in which the light from the light irradiation unit on the back surface side of the target substance detection chip is irradiated with the slide member holding the electromagnet or the permanent magnet. 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 a vector component in a direction parallel to the in-plane direction of the surface of the target substance detection chip. 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.
The magnetic particles are not particularly limited, and known magnetic beads or the like can be used.

(Target substance detection method)
The target substance detection chip of the present invention includes a target substance detection method for detecting reflected light from the electric field enhancing layer and the target substance or the target substance contained in the liquid sample held on the target substance detection chip. It can be applied to any of the target substance detection methods for detecting fluorescence or scattered light from the bound labeling substance.
The first target substance detection method of the present invention is, as the former aspect, a liquid sample introduction step of introducing the liquid sample into the closed water-repellent portion of the target substance detection chip of the present invention, and a liquid sample introduction step of the target substance detection chip. It includes a light irradiation step of irradiating the enhanced electric field layer with light from the back surface side under all reflection conditions, and a reflected light detection step of being reflected from the electric field enhancing layer.
The second target substance detection method of the present invention is, as the latter aspect, a liquid sample introduction step of introducing the liquid sample into the closed water-repellent portion of the target substance detection chip of the present invention, and a liquid sample introduction step of the target substance detection chip. A light irradiation step of irradiating the enhanced electric field layer with light from the back surface side under all reflection conditions, and fluorescence emitted from a target substance contained in the liquid sample or a labeling substance bound to the target substance based on the light irradiation. Alternatively, it includes a light detection step of detecting scattered light.

The first target substance detection method can be carried out according to the matters described for the first target substance detection device.
Further, the second target substance detection method can be carried out according to the matters described for the second target substance detection device.

Next, the target substance detection device and the first embodiment of the target substance detection method of the present invention will be described with reference to FIG. Note that FIG. 4 is an explanatory diagram showing a schematic configuration of the first embodiment.

As shown in FIG. 4, the target substance detection device 10 includes a target substance detection chip 1 (see FIGS. 1 and 2), a light irradiation unit 11, an optical prism 12, and a reflected light detection unit 13.
The light irradiation unit 11 is capable of irradiating light from the back surface side to one surface of the electric field enhancing layer under total reflection conditions, with the surface on the side where the closed water repellent portion 4 of the target substance detection chip 1 is formed as the front surface. ..
The reflected light detection unit 13 is arranged on the back surface side of the target substance detection chip 1, and is capable of detecting the reflected light reflected from the electric field enhancing layer.

In the target substance detection device 10, first, the light irradiation unit 11 irradiates the electric field enhancing layer with light L under total reflection conditions, adjusts the incident angle thereof, and specifies that the reflection characteristics change in the reflected light detection unit 13. Set to the incident angle θ of. When this specific incident angle θ is known, the target substance detection device 10 is set in advance so that light is irradiated at this incident angle θ.
Next, a liquid sample for verifying the target substance is introduced into the closed water repellent portion 4. At this time, by introducing and holding the liquid samples of different types into the plurality of closed water repellent portions 4, it is possible to detect the target substance in a multi-channel manner. Light L is irradiated so that the light from the light irradiation unit 11 is simultaneously irradiated to the plurality of closed water repellent portions 4, and the reflected light image from the plurality of closed water repellent portions 4 is transmitted by an imaging device such as an image sensor. By acquiring and observing the light and darkness, it is possible to detect the target substance in each of the closed water repellent portions 4 and determine the amount thereof at once.
When the target substance is not present in advance, the reflected light is strong, and when the target substance is present and the target substance is adsorbed on the surface of the electric field enhancing layer, the reflected light intensity is weakened. Depending on the condition, the target substance can be detected and the amount thereof can be determined.
On the contrary, when the target substance is not present in advance, the reflected light is weak, and when the target substance is present and the target substance is adsorbed on the surface of the electric field enhancing layer, the reflected light intensity is set to be strong. The target substance can be detected and the amount thereof can be determined depending on the degree of increase in strength.

Next, the second embodiment of the target substance detection device and the target substance detection method of the present invention will be described with reference to FIG. Note that FIG. 5 is an explanatory diagram showing a schematic configuration of the second embodiment.

As shown in FIG. 5, the target substance detection device 20 includes a target substance detection chip 1 (see FIGS. 1 and 2), a light irradiation unit 21, an optical prism 22, and a light detection unit 23.
The light irradiation unit 21 is configured according to the light irradiation unit 11.
The light detection unit 23 is arranged on the surface side of the target substance detection chip 1, and is a target substance contained in a liquid sample held in the closed water repellent unit 4 based on irradiation with light L, or a label bonded to the target substance. It is capable of detecting fluorescence or scattered light emitted from a substance, and is composed of an imaging device and the like.

In the target substance detection device 20, first, a liquid sample for verifying the target substance is introduced into the closed water repellent portion 4. At this time, by introducing and holding the liquid samples of different types into the plurality of closed water repellent portions 4, it is possible to detect the target substance in a multi-channel manner.
Next, the light irradiation unit 21 irradiates the electric field enhancing layer in the plurality of closed water repellent portions 4 with light L under total reflection conditions to form the enhanced electric field in the vicinity of the surface of the target substance detection chip 1.
Next, using the light enhanced by the enhanced electric field as excitation light, an optical signal S of light emitted from the target substance or a labeling substance such as a fluorescent substance or a light scattering substance that binds to and labels the target substance is used. The target substance is detected by detecting with the light detection unit 22.

Next, the third embodiment of the target substance detection device and the target substance detection method of the present invention will be described with reference to FIG. Note that FIG. 6 is an explanatory diagram showing a schematic configuration of the third embodiment.

As shown in FIG. 6, the target substance detection device 30 includes a target substance detection chip 1 (see FIGS. 1 and 2), a light irradiation unit 31, an optical prism 32, a light detection unit 33, and a magnetic field application unit 34.
The target substance detection device 30 is different from the target substance detection device 20 in that the magnetic field application unit 34 is arranged, and the light irradiation unit 31, the optical prism 32 and the light detection unit 33 are the light irradiation unit 21 in the target substance detection device 20. , The optical prism 22 and the photodetector 23 can be configured in the same manner. Hereinafter, the description focusing on the magnetic field application unit 34 will be described.

The magnetic field application unit 34 is composed of a slide member holding a permanent magnet or the like, is arranged on the back surface side of the target substance detection chip 1, and is a vector in a direction parallel to the in-plane direction of the front surface of the target substance detection chip 1. It is possible to slide and move in the direction of having the component (X ₁ and X _{2 in the figure).}
The liquid sample held in the closed water-repellent portion 4 is prepared by adding the magnetic particles forming a conjugate with the target substance.
In the target substance detection device 30 in which such a magnetic field application unit 34 is arranged, the target substance detection device 30 is targeted by applying a magnetic field by the magnetic field application unit 34 without waiting for the gravity sedimentation of the coupling body floating in the liquid sample. It can be attracted to the surface of the substance detection chip 1 and can be measured in a short time.
Further, by comparing and observing the movement of the coupled body accompanying the slide movement of the magnetic field application unit 34 before and after the slide movement, observation is performed excluding the noise signal included in the optical signal before the magnetic field is applied. be able to.
That is, while the conjugate moves by the slide movement, the noise signal caused by scratches on the surface of the target substance detection chip does not move by the slide movement, so that the noise signal moves with the slide movement. The noise signal can be eliminated by performing the detection focusing on the optical signal.

1 Target substance detection chip 2 Light transmissive substrate 3 Electric field enhancement layer 4 Closed water repellent part 5 Liquid sample 10, 20, 30 Target substance detection device 11, 21, 31 Light irradiation part 12, 22, 32 Optical prism 13 Reflected light detection Part 23, 33 Light detection part 34 Magnetic field application part

Claims (7)

Light transmissive substrate and
An electric field enhancing layer formed on the light transmissive substrate and capable of exciting either surface plasmon resonance or waveguide mode when light is irradiated from one surface under total reflection conditions.
A plurality of the electric field enhancing layers are formed on any of the surface of the light transmissive substrate on which the electric field enhancing layer is formed and the other surface of the electric field enhancing layer, and the electric field enhancing layer is formed when viewed from the other surface. The closed water repellent part of the shape drawn by the surrounding closed figure,
Is arranged ,
The target substance is characterized in that the closed figure is drawn in a regular hexagonal shape by a linear film having a width of 5 μm to 20 μm, and adjacent closed water-repellent portions are formed so as to share one side of the regular hexagon. Detection chip. A plurality of electric field enhancing layers are formed scattered on the light transmissive substrate, and closed water repellent portions are formed on the surface of the light transmissive substrate on which the electric field enhancing layer is formed. The target substance detection chip according to claim 1, wherein one electric field enhancing layer is formed in the closed figure of the closed water repellent portion so as to be separated from the drawn line of the closed figure. The target substance detection chip according to any one of claims 1 to 2, wherein the maximum diameter of the closed figure is at most 5 mm. The target substance detection chip according to any one of claims 1 to 3 and
A light irradiation unit capable of irradiating one surface of the electric field enhancing layer from the back surface side under total reflection conditions with the surface on the side where the closed water repellent portion of the target substance detection chip is formed as the front surface.
A reflected light detection unit arranged on the back surface side of the target substance detection chip and capable of detecting the reflected light reflected from the electric field enhancing layer.
A target substance detection device characterized by being arranged. The target substance detection chip according to any one of claims 1 to 3 and
A light irradiation unit capable of irradiating one surface of the electric field enhancing layer from the back surface side under total reflection conditions with the surface on the side where the closed water repellent portion of the target substance detection chip is formed as the front surface.
Fluorescence or scattering emitted from the target substance contained in the liquid sample arranged on the surface side of the target substance detection chip and held in the closed water-repellent portion based on the irradiation of light or the labeling substance bound to the target substance. A light detector that can detect light,
A target substance detection device characterized by being arranged. A target substance detection method for detecting a target substance using the target substance detection chip according to any one of claims 1 to 3.
The liquid sample introduction process of introducing the liquid sample into the closed water-repellent part of the target substance detection chip,
A light irradiation step of irradiating one surface of the electric field enhancing layer from the back surface side of the target substance detection chip under total reflection conditions, and a light irradiation step.
A reflected light detection step for detecting the reflected light reflected from the electric field enhancing layer, and
A method for detecting a target substance, which comprises. A target substance detection method for detecting a target substance using the target substance detection chip according to any one of claims 1 to 3.
The liquid sample introduction process of introducing the liquid sample into the closed water-repellent part of the target substance detection chip,
A light irradiation step of irradiating one surface of the electric field enhancing layer from the back surface side of the target substance detection chip under total reflection conditions, and a light irradiation step.
A light detection step of detecting fluorescence or scattered light emitted from a target substance contained in the liquid sample or a labeling substance bound to the target substance based on the irradiation of the light.
A method for detecting a target substance, which comprises.

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