JP2018179785A - Target substance detection chip, target substance detection device and target substance detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a multi-channel type target substance detection chip which 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. A target substance detection chip of the present invention is formed on a light transmissive substrate 2 and a light transmissive substrate 2, and surface plasmon resonance and waveguide are formed when light is irradiated from one surface under total reflection conditions. A plurality of electric field enhancing layers 3 that can be excited by the wave mode, a surface on which the electric field enhancing layer 3 of the light transmissive substrate 2 is formed, and another surface of the electric field enhancing layer 3 are formed. It is characterized in that a closed water-repellent portion 4 having a shape drawn by a closed figure surrounding the electric field enhancing layer 3 when viewed from another surface is arranged. [Selection diagram] Fig. 2

Description

  The present invention relates to a target substance detection chip, a target substance detection device, and a target substance detection method capable of simultaneously holding a plurality of liquid samples and performing independent observation in optical observation using surface plasmon resonance and a waveguide mode.

  Recently, in various fields such as medical checkup, medicine, early detection of diseases and infectious diseases, environmental pollution detection, anti-terrorism, etc., a portable, easy-to-use and highly sensitive detector is required. SPR sensors that use surface plasmon resonance (SPR) and waveguide sensors that use a waveguide mode are known as sensors that are small enough to be carried and that can measure various substances contained in liquid. (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, selective detection of various biomaterials such as proteins, assessment of environmental contamination, and detection of poisons, illegal drugs and explosives used for terrorism It has been used for

As a SPR sensor, a Kretschmann placement SPR sensor is widely used.
In the Kretschmann configuration type SPR sensor, a metal thin film layer is formed by vapor-depositing a metal such as gold, silver, or aluminum on a transparent substrate, and the optical thin film is formed on the surface of the transparent substrate opposite to the surface on which the metal thin film layer is formed. The laser light emitted from the light source is polarized by a polarizing plate, and the transparent substrate is irradiated via the optical prism. Incident light is incident under the condition of total reflection. The surface plasmon resonance is expressed at a specific incident angle by the evanescent wave that exudes to the surface side of the metal thin film layer of the incident light. When the surface plasmon resonance occurs, the evanescent wave is absorbed by the surface plasmon, so that the intensity of the reflected light is significantly reduced near this incident angle. The conditions under which the surface plasmon resonance appears are changed by the dielectric constant in the vicinity of the surface of the metal thin film layer, so if adsorption, approach, detachment, or deterioration of a substance occurs on the surface of the metal thin film layer, the intensity of the reflected light is A change appears. Therefore, when the target substance is bonded or adsorbed on the surface of the metal thin film layer to change the dielectric constant, the reflection characteristic of the incident light changes, so that the reflected light reflected from the metal thin film layer The target substance can be detected by monitoring the intensity change with a photodetector.

The waveguide mode sensor has a structure similar to that of the SPR sensor, and is a sensor that detects the adsorption of a substance on the detection surface and the change in the dielectric constant. 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 Kretschmann arrangement, a transparent substrate, a thin film layer composed of a metal layer or a semiconductor layer coated thereon, and a dielectric layer formed on the thin film layer Use a detection plate consisting of An optical prism is in close contact with the surface of the detection plate opposite to the surface on which the dielectric layer is formed, via a refractive index adjustment oil. The incident light emitted from the light source and polarized by the polarizing plate is irradiated to the detection plate through the optical prism. The incident light is incident on the detection plate under the condition of total reflection. At a particular angle of incidence, when the incident light combines with a guided mode (also called leaky mode or leaky mode) propagating in the dielectric layer, the guided mode is excited and light near the incident angle is The reflected light intensity changes greatly. Since the excitation condition of such a waveguide mode changes depending on the dielectric constant in the vicinity of the surface of the dielectric layer, when adsorption, approach, detachment, or alteration of the target substance occurs on the surface of the dielectric layer, the reflected light is A change appears in the intensity of By observing this change with a photodetector, phenomena such as adsorption, approach, detachment, or deterioration of the target substance can be detected.

In addition, the SPR sensor and the waveguide mode sensor also have an effect of enhancing the light emission of the fluorescent substance if a fluorescent substance (for example, a fluorescent dye or the like) capable of photoexcitation emission is attached to or close to the detection surface. That is, when light is irradiated to one surface of the detection plate under total reflection conditions, an enhanced electric field is formed on the other surface, and the fluorescent material can be made to emit light strongly by using the enhanced electric field as excitation light. It is possible to perform fluorescence observation with less background light (see, for example, Patent Document 8).
When a light scattering material (for example, nanoparticles composed of a scatterer such as polystyrene beads or gold nanoparticles) is attached to or in proximity to the detection surface of the SPR sensor or the waveguide mode sensor, the light scattering material It also has the effect of enhancing scattered light. That is, when light is irradiated to one surface of the detection plate 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 material. And scattered light observation with high brightness can be performed (see, for example, Non-Patent Document 20).
Various methods have been proposed as methods for producing electric field enhancement by total reflection of light and obtaining an enhanced electric field, and the present inventor has developed 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 under the condition of total reflection on the surface of the detection plate through the prism to obtain an enhanced electric field (see Non-Patent Document 21).

By the way, the present inventor simultaneously holds a plurality of liquid samples for the purpose of reducing the operation load when using the SPR sensor or the guided mode sensor and performing efficient observation, and independent observation. Patent Document 9 proposes a multi-channel type detection plate capable of performing the following.
However, in the proposal of this multi-channel type detection plate, in order to introduce the liquid sample in the vicinity of the surface of the detection chip, a concave accommodation portion for accommodating the detection chip and a flow for sending the liquid sample to the detection chip There is a need to make structures that do not directly participate in the formation of the enhanced electric field, such as vias.
Therefore, at present, there is a demand for development of a new detection chip which can be manufactured in a smaller size and at low cost by reducing the number of parts.

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

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  The present invention solves the above problems in the prior art, and provides a multi-channel target substance detection chip which 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. It will be an issue.

The means for solving the problems are as follows. That is,
<1> A light transmitting substrate, and an electric field enhancing layer formed on the light transmitting substrate and capable of exciting surface plasmon resonance and a waveguide mode when light is irradiated from one side under total reflection conditions And a plurality of surfaces formed on the surface of the light transmitting substrate on which the electric field enhancing layer is formed and any of the other surfaces of the electric field enhancing layer, the electric field enhancing when viewed from the other surface A target substance detection chip characterized in that a closed water repellent portion having a shape drawn by a closed figure surrounding a layer is disposed.
<2> The target substance detection chip according to <1>, wherein the closed figure has a polygonal shape, and the adjacent closed water repellent portions are formed so as to share one side of the polygonal shape.
<3> Any one of the above <1> to <2>, wherein the closed figure is any one of an equilateral triangle, an isosceles triangle, a right triangle, a rhombus, a parallelogram, a rectangle, a square, an equilateral hexagon and an octagon. Target substance detection chip.
<4> A plurality of electric field enhancing layers are formed dotted on the light transmitting substrate, and a closed water repellent portion is formed on the surface of the light transmitting substrate on which the electric field enhancing layer is formed, Also, the target substance detection chip according to any one of <1> to <3>, wherein one of the electric field enhancing layers is formed in a closed shape of the closed water repellent portion at a distance from a drawing line of the closed shape.
<5> The target substance detection chip according to any one of <1> to <4>, wherein the maximum diameter of the closed figure is at most 5 mm.
<6> The target substance detection chip according to any one of <1> to <5>, and the surface on the side on which the closed water repellent part of the target substance detection chip is formed is the front surface side of the electric field enhancing layer. A light irradiator capable of emitting light under total reflection conditions on one surface, and a reflection that is disposed on the back surface side of the target substance detection chip and is capable of detecting reflected light reflected from the electric field enhancing layer A target substance detection device comprising: a light detection unit.
<7> The target substance detection chip according to any one of <1> to <5>, and the surface on the side on which the closed water repellent part of the target substance detection chip is formed is the front surface side of the electric field enhancing layer. A light irradiator capable of emitting light on one surface under total reflection conditions, and a liquid sample disposed 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 target substance contained in or a light detection unit capable of detecting fluorescence or scattered light emitted from a labeling substance bound to the target substance is disposed.
<8> A target substance detection method for detecting a target substance using the target substance detection chip according to <1> to <5>, wherein the liquid introduces a liquid sample into the closed water repellent portion of the target substance detection chip A sample introduction step, a light irradiation step of irradiating light from the back surface side of the target substance detection chip to one surface of the electric field enhancing layer under total reflection conditions, reflected light to detect reflected light reflected from the electric field enhancing layer And a detection step.
<9> A target substance detection method for detecting a target substance using the target substance detection chip according to <1> to <5>, wherein the liquid introduces a liquid sample into the closed water repellent portion of the target substance detection chip The target substance contained in the liquid sample based on the sample introduction step, the light irradiation step of irradiating light under total reflection conditions to one surface of the electric field enhancing layer from the back surface side of the target substance detection chip, and the irradiation of the light And d) detecting the fluorescence or scattered light emitted from the labeling substance bound to the target substance.

  According to the present invention, the aforementioned problems in the prior art can be solved, and a small-sized, low-cost, multi-channel target substance detection chip that can be manufactured 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 schematic structure of the target substance detection chip which concerns on one Embodiment of this invention. It is explanatory drawing which shows a mode when one part of the target substance detection chip | tip shown in FIG. 1 is expanded and seen from the other surface of an electric field enhancement layer. FIG. 5 is an enlarged cross-sectional view of a structure corresponding to one closed water repellent portion 4; It is an explanatory view showing a schematic structure of a 1st embodiment. It is explanatory drawing which shows schematic structure of 2nd Embodiment. It is explanatory drawing which shows 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 transmission 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.
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.
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 capable of exciting either of surface plasmon resonance and a waveguide mode when light is irradiated from one side 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 observing emission from a target substance or a fluorescent substance that labels the target substance, when the target substance and the fluorescent substance are in proximity to the metal layer, the energy obtained from the excitation light by the target substance or the fluorescent substance is A phenomenon called "quenching" may occur in which the metal layer migrates and the light emission efficiency decreases.
In this case, if 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 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 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.
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.

<Closed water repellent part>
The closed water repellent portion is formed in a plurality on the surface of the light transmitting substrate on which the electric field enhancing layer is formed and on the other surface of the electric field enhancing layer, and viewed from the other surface. When it is drawn, it is drawn as a closed figure surrounding the electric field enhancing layer.

As mentioned 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 can be formed on the light transmitting substrate, and the electric field enhancing layer can be formed in the closed pattern of the closed water repellent portion. The order of formation is not particularly limited, and the electric field enhancing layer may be formed after forming the closed water repellent portion, or the closed water repellent portion may be formed after forming the electric field enhancing layer. .
In the latter case, the electric field enhancing layer may be formed on the light transmitting substrate and then the closed water repellent portion may be formed on the electric field enhancing layer.

  There is no restriction | limiting in particular as a formation method of the said closed water repellent part, According to the objective, it can select suitably, For example, vapor deposition of the linear film of the well-known water repellent compound of fluorine type, silicone type, and acryl type is vapor-deposited. It can be formed by a known forming method such as a method, a sputtering method, a coating method, and a printing method. In addition, a known fluorine coating method in which a known fluorine-based water repellent gas is subjected to plasma irradiation may be mentioned.

In the case of forming the closed water repellent portion by the printing method as an example, there is no particular limitation, but known water repellent properties including a silicone type surfactant or a fluorine type surfactant together with a binder resin, a solvent, a reactive diluent and the like Ink can be used.
There is no restriction | limiting in particular as content of said each surfactant in the said water-repellent ink, It is about 0.5 mass%-20 mass%.

The silicone surfactant is not particularly limited and may be appropriately selected from known ones. For example, silicone oils having a dimethylsiloxane skeleton, silicone resins, and some of the methyl groups of these silicones are alkyl. A modified silicone oil substituted by a group, an aryl group, an alkoxy group, a hydroxyl group etc., a modified silicone resin etc. are mentioned.
Moreover, there is no restriction | limiting in particular also as said fluorine-type surfactant, It can select suitably from well-known things, can be used, for example, the monomer which has a perfluoroalkyl group, and the monomer which has various reactive groups are made to react. And polymers and oligomers having a perfluoroalkyl group in the side chain.

The binder resin is not particularly limited, and may be arbitrarily selected from polymer compounds which are compatible with the silicone surfactant or the fluorosurfactant and become solid after drying.
Examples of the polymer compound include cellulose resins such as acrylic resin, epoxy resin, melamine resin, urethane resin, polyurethane resin, polyester resin, butyral resin, polyvinyl alcohol resin, acetal resin, phenol resin, nitrocellulose, and ethyl cellulose; Chlorinated rubber, petroleum resin, vinylidene fluoride resin and the like can be mentioned.

As the drawing (patterning) method using the water repellent ink, any printing method can be adopted, but when the pattern accuracy is about 5 μm to 20 μm width, the letterpress reverse printing method is preferable, and the pattern accuracy is When it exceeds 20 μm, a gravure printing method, a gravure offset printing method, a flexographic 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 flat figure without an 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, for example, a polygonal shape, a circular shape, or an elliptical shape. Can. Among these, the polygonal shape is preferable.
When the closed figure is the polygonal shape, adjacent closed water repellent portions are formed so as to share one side of the polygonal shape, and many closed water repellent portions are formed in a limited area. It is possible to reduce the size while securing the necessary number of channels.
Furthermore, when the polygonal shape is any one of an equilateral triangle, an isosceles triangle, a right triangle, a rhombus, a parallelogram, a rectangle, an equilateral tetragon, an equilateral hexagon and an octagon, the plurality of closed water repellents in a limited area The parts can be arranged in close line, and more closed water repellent parts can be formed.
The polygonal shape means a shape drawn by connecting straight lines with a maximum internal angle of less than 180 ° and a length of at least 1 μm.

  The size of the closed water repellent portion is not particularly limited, and depending on the spot diameter of the light irradiated to the one surface of the electric field enhancing layer, the smaller the number, the larger the number of the above-mentioned regions. A closed water repellent portion can be formed, and the maximum diameter of the closed figure is preferably at most 5 mm.

Hereinafter, an example of an 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. FIG. 2 is an explanatory view showing a part of the target substance detection chip shown in FIG. 1 in an enlarged view from 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 transmitting substrate 2, a plurality of electric field enhancing layers 3 and a plurality of closed water repellent portions 4.
In the target substance detection chip 1, the plurality of electric field enhancing layers 3 are formed on the light transmitting substrate 2 so as to be dotted, and the closed water repellent portion 4 is formed as the electric field enhancing layer 3 of the light transmitting substrate 2. An electric field enhancing layer 3 is formed on the closed surface of the closed water repellent portion 4 at a distance from the drawing line of the closed shape.

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

The smaller the maximum diameter L ₁ of the closed water repellent portion 4 in the closed figure, the larger number of the closed water repellent portions 4 can be formed in a limited area, and it is preferably at most 5 mm.
In addition, the electric field enhancing layer 3 is disposed at the central position of the closed figure so that one electric field enhancing layer 3 is formed apart from the drawing line of the closed figure in the closed figure of the closed water repellent portion 4. And the maximum diameter L ₂ is preferably smaller than L ₁ .

The appearance of the liquid sample introduced into the target substance detection chip 1 is shown in FIG. FIG. 3 is a cross-sectional view showing a structure corresponding to one closing water repelling part 4 in an enlarged manner.
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 contained in the closed water repellent portion 4 due to the water repellency of the closed water repellent portion 4 as shown in FIG. It is held.
Therefore, the target substance detection chip 1 is a multichannel detection chip capable of simultaneously holding different types of liquid samples 5 by the plurality of closed water repellent portions 4 and capable of observing each liquid sample 5 independently.

  In the target substance detection chip 1 configured in this manner, it is not necessary to produce a structure that does not directly participate in the formation of the enhanced electric field, such as the concave housing portion or the flow path in the prior art, and the number of parts can be reduced. It can be manufactured at small size and at low cost.

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

<Light irradiator>
The light irradiation unit is capable of irradiating light from the back surface side to one surface of the electric field enhancing layer under the condition of total reflection, with the surface on the side on which the closed water repellent portion of the target substance detection chip is formed as the surface. Be done.
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.

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

  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. The object is such that the light introduced into the target substance detection chip is irradiated with the light introduced into the target substance detection chip under total reflection conditions to form the enhanced electric field on the surface. The 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.

<Reflected light detection unit>
The reflected light detection unit is disposed on the back surface side of the target substance detection chip, and is capable of detecting reflected light reflected from the electric field enhancing layer.
The reflected light detection unit is not particularly limited, and can be configured using a known light detection device 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. When used, a plurality of closed water repellent portions 4 can be simultaneously observed, which is preferable.

In the first target substance detection device, when the electric field enhancing layer is formed of the surface plasmon excitation layer, the light transmitting substrate is irradiated with light on the one surface of the electric field enhancing layer under total reflection conditions. Evanescent light exudes from the surface 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 part is adjusted, surface plasmon resonance is exhibited in which the evanescent light is absorbed by the surface plasmon at a specific incident angle θ, and the reflected light intensity from the electric field enhancing layer is It decreases significantly. The reflected light intensity is detected at each incident angle by the reflected light detection unit.
Since the incident angle θ shifts and changes according to the change of the dielectric constant in the vicinity of the surface, the target substance in the liquid sample held on the surface can be detected through the shift change of the incident angle θ. it can.
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 is changed according to the change of the dielectric constant near the surface. Changes occur, and changes in light and dark are observed. Therefore, the target substance in the liquid sample held on the surface can also be detected through this change in brightness.

In the first target substance detection device, when the electric field enhancement layer is formed of the guided wave mode excitation layer, the thin film layer is made to be incident light at a specific incident angle θ while satisfying the total reflection condition. And a guided mode in which propagating light propagates in the dielectric layer is excited. In the vicinity of the incident angle θ, the other incident angles and the reflected light intensity are largely different. Further, the excitation condition of the waveguide mode is sensitive to the change of the dielectric constant on the surface of the target substance detection chip, and when the dielectric constant changes due to the adsorption of the target substance or the like, It appears as fluctuations in the reflection characteristics of the reflected light.
At this time, the reflected light detecting section catches, for example, the adsorption of the target substance at the specific light incident angle θ at the time of incidence of monochromatic light by the reflected light detection unit, thereby holding the object on the surface. The target substance in the liquid sample can be detected.
In addition, 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 changed according to the change of the dielectric constant near the surface. Changes in intensity occur and changes in light and dark are observed. Therefore, the target substance in the liquid sample held on the surface can also 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 and configured from the items described for the first target substance detection device.

<Light detection unit>
The light detection unit is disposed on the surface side of the target substance detection chip, and the target substance included in the liquid sample or the label bound to the target substance is held in the closed water repellent portion based on the light irradiation. The fluorescent or scattered light emitted from the substance can be detected.
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, a plurality of closed water repellent portions 4 can be simultaneously observed, and further, position information of the optical signal in the two-dimensional image information appearing as a light spot, It is possible to observe size information observed in a dimension and increase / decrease information of light signal intensity at a light spot in time series. 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.

The labeling substance is used to label the target substance when the target substance is a substance that hardly generates fluorescence or scattered light.
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.

  In the second target substance detection device, the second target substance detection device is formed in the vicinity of the other surface of the electric field enhancing layer (the surface of the target substance detection chip) based on excitation of either the surface plasmon resonance or the waveguide mode. The target substance or a fluorescent substance for labeling the target substance is caused to emit light by using the enhanced electric field as excitation light, or scattered light is generated from a light scattering substance for labeling the target substance or the target substance, and the light A 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, since the liquid sample exhibits an upward convex shape by surface tension as shown in FIG. 3, a lens effect works, and when the imaging device is used for the light detection unit, the target substance detection chip When the surface is observed, defocusing occurs, making it difficult to obtain an image. Therefore, it is preferable to dispose a cover glass on the liquid sample to flatten the liquid surface.

By the way, in the second target substance detection device, the liquid sample is introduced into the closed water repellent portion in order to use the enhanced electric field formed in the vicinity of the surface of the target substance detection chip. It is necessary to wait for the target substance floating in the inside to fall near the surface of the target substance detection chip by gravity.
Therefore, when performing measurement 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 that floats in the liquid sample is the above-mentioned target substance detection chip It is effective to apply a magnetic field that attracts to the surface.
Therefore, it is preferable that a magnetic field application unit capable of applying such a magnetic field is further disposed as the second target substance detection device. Specifically, it is preferable that a magnetic field application unit capable of applying a magnetic field for attracting the combined body to the surface of the target substance detection chip is disposed 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 light signal scatters light due to dirt or scratches on the surface of the target substance detection chip, the target substance The detection accuracy is lowered if noise signals based on, for example, auto-fluorescence generated from the components of the detection chip and light emission from impurities contained in the liquid sample are included.
From the above, the state of the combined body in which the magnetic particles are bound to the target substance is compared and observed before and after the application of the magnetic field by the magnetic field application unit using the light detection unit to obtain an optical signal before the magnetic field application. It is effective to perform the observation excluding the included noise signal.
That is, while the combined body is moved by the application of the magnetic field, the noise signal caused by a flaw or the like on the surface of the target substance detection chip is not moved by the application of the magnetic field. The noise signal can be eliminated by performing detection based on the moving optical signal.
Therefore, it is preferable that a magnetic field application unit capable of applying such a magnetic field is further disposed as the second target substance detection device. Specifically, it is possible to apply a first magnetic field that moves the combined body contained in the liquid sample held on 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 possible to apply a second magnetic field that attracts the combined substance in the liquid sample that is disposed on the back surface side of the target substance detection chip and is held on the surface, And applying a magnetic field of any of the second magnetic field application units 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 with the second magnetic field applied. Preferably, parts are arranged. Among them, it is particularly preferable to dispose the second magnetic field application unit because the attraction magnetic field for the purpose of measurement in a short time can be applied.

In addition, there is no restriction | limiting in particular as a component of the said magnetic field application part, A well-known permanent magnet, an electromagnet, etc. can be mentioned. In addition, as the second magnetic field application unit, an area to which the light from the light irradiation unit on the back surface side of the target substance detection chip is irradiated (the detection is performed on the slide member holding the electromagnet or the permanent magnet) The electromagnet or permanent magnet in a direction having an initial state in which the electromagnet or the permanent magnet is positioned near the region and 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 performing movement control between the moved state. 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.
Moreover, there is no restriction | limiting in particular as said magnetic particle, A well-known magnetic bead etc. can be used.

(Target substance detection method)
The target substance detection chip according to the present invention includes a target substance detection method for detecting reflected light from the electric field enhancing layer, a target substance contained in the liquid sample held on the target substance detection chip, or the target substance. The present invention can be applied to any target substance detection method for detecting fluorescence or scattered light from a bound label substance.
In the first target substance detection method of the present invention, a liquid sample introducing step of introducing the liquid sample into the closed water repellent portion of the target substance detection chip of the present invention as a mode of the former; It includes a light irradiation step of irradiating light from the back side to the enhanced electric field layer under total reflection conditions, and a reflected light detection step reflected from the electric field enhancing layer.
In the second target substance detection method of the present invention, 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; A light irradiation step of irradiating light from the back side to the enhanced electric field layer under total reflection conditions; fluorescence emitted from the target substance contained in the liquid sample or the labeled substance bound to the target substance based on the irradiation of the light Or a light detection step of detecting scattered light.

The first target substance detection method can be implemented according to the items described for the first target substance detection device.
The second target substance detection method can be implemented according to the items described for the second target substance detection device.

  Next, a first embodiment of the target substance detection device and the target substance detection method of the present invention will be described with reference to FIG. FIG. 4 is an explanatory view 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 irradiator 11 can emit light under total reflection conditions from the back surface side to the surface of the electric field enhancing layer with the surface on the side on which the closed water repellent portion 4 of the target substance detection chip 1 is formed as the surface. .
The reflected light detection unit 13 is disposed 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, while irradiating the light L from the light irradiation unit 11 to the electric field enhancing layer under total reflection conditions, the incident angle is adjusted, and the reflection characteristic is changed in the reflected light detection unit 13 It is set to the incident angle θ of When the specific incident angle θ is known, the target substance detection device 10 is set in advance so that light is emitted at the incident angle θ.
Next, a liquid sample to be tested for the target substance is introduced into the closed water repellent portion 4. At this time, by introducing the different types of liquid samples into the plurality of closed water repellent portions 4 and holding them, it is possible to detect the multi-channeled target substance. The 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 by an imaging device such as an image sensor Is obtained, and detection of the target substance in each of the closed water repellent portions 4 and determination of the amount thereof can be performed at once.
When the target substance is not present, 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 intensity of the reflected light is attenuated when it is set to be weak. Depending on the condition, detection of the target substance and determination of its amount can be performed.
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 increased. Detection of the target substance and determination of its amount can be performed according to the degree of increase in intensity.

  Next, a 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. FIG. 5 is an explanatory view showing a schematic configuration of the second embodiment.

As shown in FIG. 5, the target substance detection device 20 includes the 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 disposed on the surface side of the target substance detection chip 1 and is bound to the target substance contained in the liquid sample held in the closed water repellent part 4 based on the irradiation of the light L or the target substance It is possible to detect fluorescence or scattered light emitted from a substance, and is configured by an imaging device or the like.

In the target substance detection device 20, first, a liquid sample to be tested for the target substance is introduced into the closed water repellent portion 4. At this time, by introducing the different types of liquid samples into the plurality of closed water repellent portions 4 and holding them, it is possible to detect the multi-channeled target substance.
Next, light L is irradiated from the light irradiation part 21 to the electric field enhancing layer in the plurality of closed water repellent parts 4 under total reflection conditions to form the enhanced electric field in the vicinity of the surface of the target substance detection chip 1.
Next, the light signal S of light emitted from a labeling substance such as a fluorescent substance or a light scattering substance which is bound to the target substance or the target substance and labeled by using light enhanced by the enhanced electric field as excitation light The target substance is detected by the light detection unit 22.

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

As shown in FIG. 6, the target substance detection device 30 includes the 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 differs from the target substance detection device 20 in that the magnetic field application unit 34 is disposed, and the light irradiation portion 31, the optical prism 32 and the light detection portion 33 are the light irradiation portion 21 in the target material detection device 20. The optical prism 22 and the light detection unit 23 can be configured in the same manner. The following description will focus on the magnetic field application unit 34.

The magnetic field application unit 34 is formed of a slide member holding a permanent magnet or the like, and is disposed on the back side of the target substance detection chip 1 and is a vector in a direction parallel to the in-plane direction of the surface of the target substance detection chip 1 It is possible to slide in a direction (X ₁ and X ₂ in the figure) having the component.
The liquid sample held in the closed water repellent portion 4 is prepared by adding the magnetic particles constituting a conjugate with the target substance.
In the target substance detection device 30 in which the magnetic field application unit 34 is disposed, the combination object is aimed by applying the magnetic field by the magnetic field application unit 34 without waiting for the gravity settling of the combined object floating in the liquid sample. It can be drawn to the surface of the substance detection chip 1 and measurement can be made in a short time.
In addition, by comparing and observing the state of movement of the combined body with the slide movement of the magnetic field application unit 34 before and after the slide movement, observation is performed in which the noise signal included in the light signal before the magnetic field application is eliminated. be able to.
That is, while the combined body is moved by the slide movement, the noise signal caused by a flaw or the like on the surface of the target substance detection chip is not moved by the slide movement, so it moves along with the slide movement. The noise signal can be eliminated by performing detection based on the light signal.

DESCRIPTION OF SYMBOLS 1 target substance detection chip 2 light transmitting substrate 3 electric field enhancing 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 Section 23, 33 Light detection section 34 Magnetic field application section

Claims (9)

A light transmitting substrate,
An electric field enhancing layer formed on the light transmitting substrate, wherein surface plasmon resonance and a waveguide mode can be excited when light is irradiated from one side under total reflection conditions;
A plurality is formed on the surface of the light transmitting substrate on which the electric field enhancing layer is formed and any of the other surfaces of the electric field enhancing layer, and the electric field enhancing layer is viewed from the other surface A closed water repellent portion having a shape drawn by a surrounding closed shape;
A target substance detection chip characterized in that   The target substance detection chip according to claim 1, wherein the closed figure is a polygonal shape, and adjacent closed water repellent portions are formed so as to share one side of the polygonal shape.   The target substance detection chip according to any one of claims 1 to 2, wherein the closed figure is any one of an equilateral triangle, an isosceles triangle, a right triangle, a rhombus, a parallelogram, a rectangle, a square, an equilateral hexagon and an octagon.   A plurality of electric field enhancing layers are formed on the light transmitting substrate in a scattered manner, and a closed water repellent portion is formed on the surface of the light transmitting substrate on which the electric field enhancing layer is formed, and The target substance detection chip according to any one of claims 1 to 3, wherein one of the electric field enhancing layers is formed apart from the drawing line of the closed figure in the closed figure of the closed water repellent part.   5. The target substance detection chip according to any one of claims 1 to 4, 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 5,
A light irradiator capable of emitting light under total reflection conditions to one surface of the electric field enhancing layer from the back side with the surface on the side on which the closed water repellent portion of the target substance detection chip is formed as the surface;
A reflected light detection unit disposed on the back surface side of the target substance detection chip and capable of detecting reflected light reflected from the electric field enhancing layer;
The target substance detection device characterized in that The target substance detection chip according to any one of claims 1 to 5,
A light irradiator capable of emitting light under total reflection conditions to one surface of the electric field enhancing layer from the back side with the surface on the side on which the closed water repellent portion of the target substance detection chip is formed as the surface;
The fluorescence or scattering emitted from the target substance contained in the liquid sample held in the closed water repellent part on the surface side of the target substance detection chip and held on the closed water repellant part upon irradiation of the light or the labeling substance bound to the target substance A light detection unit capable of detecting light;
The target substance detection device characterized in that A target substance detection method for detecting a target substance using the target substance detection chip according to any one of claims 1 to 5,
A liquid sample introduction step of introducing a liquid sample into the closed water repellent part of the target substance detection chip;
A light irradiation step of irradiating light from the back surface side of the target substance detection chip to one surface of the electric field enhancing layer under total reflection conditions;
A reflected light detection step of detecting reflected light reflected from the electric field enhancing layer;
A target substance detection method characterized by including. A target substance detection method for detecting a target substance using the target substance detection chip according to any one of claims 1 to 5,
A liquid sample introduction step of introducing a liquid sample into the closed water repellent part of the target substance detection chip;
A light irradiation step of irradiating light from the back surface side of the target substance detection chip to one surface of the electric field enhancing layer under total reflection conditions;
A light detection step of detecting fluorescence or scattered light emitted from a target substance contained in the liquid sample or a labeled substance bound to the target substance based on the irradiation of the light;
A target substance detection method characterized by including.