JP2019035683A - Target substance detection chip, target substance detection device, and target substance detection method - Google Patents

Abstract

To provide a target substance detection chip, a target substance detection device, and a target substance detection method capable of detecting not only an electrode but also its periphery as a detection region and detecting a target substance with high sensitivity. A target substance detection chip (1) includes a light-transmitting substrate (2) and a first layer (3) formed on the light-transmitting substrate (2) by any one of a metal material, a semiconductor material, and a first dielectric material. The second layer 4 formed of the second dielectric material is stacked in this order, and the waveguide mode excitation layer 5 that can excite the waveguide mode, and on the waveguide mode excitation layer 5 The SPR excitation layers 6a and 6b (electrodes) that can be excited by SPR are arranged so that a part of the waveguide mode excitation layer 5 is exposed. Near-field light can be formed on the exposed surfaces of the waveguide mode excitation layer 5 and the SPR excitation layers 6a and 6b by light irradiated from the back side under the total reflection condition with the surface as the back surface. And [Selection] Figure 1

Description

  The present invention detects a target substance using near-field light accompanying SPR excitation and waveguide mode excitation, and also uses a SPR excitation layer for the SPR excitation as an electrode, a target substance detection chip The present invention relates to an apparatus and a target substance detection method.

  In recent years, methods for detecting and quantifying minute substances present in a solution, in particular, biologically related substances such as DNA, RNA, proteins, viruses, and bacteria have been developed. As such a method, for example, a method using an evanescent field due to total reflection, a method using excitation of surface plasmon resonance (SPR), a waveguide mode (optical waveguide mode, waveguide mode, optical waveguide) A method using excitation of a mode (also called a mode) is known.

  As a method of using the evanescent field by total reflection, a total reflection illumination fluorescence microscope can be mentioned. The total reflection illumination fluorescence microscope is a technology that makes incident light totally reflected at the interface between the sample and the cover glass or slide glass, and uses the evanescent field generated by this as excitation light to perform fluorescence observation with less background light. (See Patent Document 1).

As a method using the SPR excitation, for example, surface plasmon resonance excitation enhanced fluorescence spectroscopy is known.
This method uses an optical arrangement called a Kretschmann arrangement to excite SPR on the metal thin film by total reflection of incident light at the interface between the metal thin film on the glass surface in contact with the prism and the liquid sample. An enhanced electric field is formed near the surface of the thin film. According to this method, the light enhanced in the vicinity of the surface of the metal thin film by excitation of the SPR is used as excitation light to excite fluorescent molecules existing in the enhanced electric field, thereby generating strong fluorescence, and background light is generated. Little fluorescence observation can be performed (see, for example, Patent Document 2).

Further, in the method using the excitation of the waveguide mode, a detection chip in which a silicon layer (semiconductor layer) and a SiO ₂ layer are laminated in this order on a silica glass substrate is placed on a trapezoidal prism made of silica glass. Then, the enhanced electric field is obtained by irradiating light from the trapezoidal prism side under the condition of total reflection on the surface of the detection chip (see Non-Patent Document 1). In this method, when the light is irradiated at a specific incident angle while satisfying the total reflection condition from the back surface side (silica glass substrate side) with respect to the detection chip, light of a specific wavelength propagates in the detection chip. It combines with the waveguide mode and utilizes the fact that the waveguide mode is excited. When the waveguide mode is excited, the enhanced electric field is generated in the vicinity of the detection chip surface. Thereby, the fluorescent molecules present in the enhanced electric field are excited, and fluorescence observation with little background light can be performed (see Non-Patent Document 2). The semiconductor layer may be formed of a metal layer, and the waveguide mode excited in a detection chip in which the semiconductor layer is formed of the metal layer may be called a leaky mode, a leakage mode, or the like. (Refer nonpatent literature 3).

By the way, in the method using the excitation of the SPR, since the formed enhanced electric field reaches only a limited region near the surface of the metal thin film, the metal thin film for the excitation of the SPR is used as an electrode. Thus, there is a technique for optically detecting the target substance on the electrode by excitation of the SPR after attracting and concentrating the target substance in the liquid sample on the electrode using electrophoresis or dielectrophoresis. It is known (for example, refer to Patent Documents 3 to 5 and Non-Patent Document 4).
However, even if the target substance is attracted onto the electrode, not all of the target substance necessarily gets onto the electrode. When the dielectrophoresis is used, depending on the frequency of the applied voltage, the target substance may be attracted not between the electrodes but between the two electrodes constituting the electrode pair.
That is, the method has a problem that the target substance cannot be detected around the electrode.

On the other hand, as a method for detecting the target substance in the periphery of the electrode, a method of performing fluorescence observation of the target substance using the evanescent field by totally reflecting light with a glass substrate located in a region between electrode pairs Has been proposed (see Patent Document 6). According to this proposal, the target substance between the electrode pairs can be detected.
However, this proposal has a problem that the target substance on the electrode cannot be detected because the electrode pair does not excite the SPR. In addition, since the method using the evanescent field due to total reflection has lower sensitivity than the method using the excitation of the SPR and the method using the waveguide mode, the proposal proposes that the target substance has high sensitivity. There is a problem that cannot be detected.

JP 2002-236258 A International Publication No. 2015/194663 JP-A-9-257702 JP 10-307104 A JP 2005-195397 A JP 2005-214890 A

M. Fujimaki et al. Optics Express, Vol. 23 (2015) pp.10925-10937 K. Nomura et al. J. Appl. Phys. Vol. 113, (2013) pp.143103-1-143103-6 R. P. Podgorsek, H. Franke, J. Woods, and S. Morrill, Sensor. Actuat. B51 pp.146-151 (1998) C. Kuroda et al., 5th International Conference on Bio-Sensing Technology, P114, Riva del Garda, Italy (2017)

  The present invention solves the above-mentioned problems in the prior art, a target substance detection chip, a target substance detection device, and a target substance capable of detecting not only the electrode but also its periphery as a detection region and detecting the target substance with high sensitivity It is an object to provide a detection method.

Means for solving the problems are as follows. That is,
<1> A light-transmitting substrate, and a first layer and a second dielectric material formed of any one of a metal material, a semiconductor material, and a first dielectric material on the light-transmitting substrate. A waveguide mode excitation layer that is configured by stacking the second layer in this order so that the waveguide mode can be excited, and a part of the waveguide mode excitation layer is exposed on the waveguide mode excitation layer. The SPR excitation layer that is configured to be laminated in a state where SPR can be excited is arranged, and the light that is irradiated under the total reflection condition from the back surface side with the surface on the light transmissive substrate side as the back surface, A target substance detection chip, wherein near-field light can be formed on the exposed surface of the waveguide mode excitation layer and the SPR excitation layer.
<2> The target substance detection chip according to <1>, wherein the SPR excitation layers are arranged as two or more layers separated from each other.
<3> The object according to any one of <1> to <2>, wherein the difference between the peak positions of the wavelengths of near-field light generated on the surfaces of the waveguide mode excitation layer and the SPR excitation layer is at most 40 nm. Substance detection chip.
<4> The target substance detection chip according to any one of <1> to <3>, wherein a material for forming the SPR excitation layer is any one of Au, Ag, and Al.
<5> The material for forming the first layer is any one of Si, Ge, SiGe, and TiO ₂ , and the material for forming the second layer is any one of SiO ₂ , PMMA, and BK7 glass <1> To <4>.
<6> Further, a constituent part of the liquid sample tank is provided on the surface of these layers so as to surround the whole or a part of the exposed waveguide mode excitation layer and the SPR excitation layer, and the surface is the bottom. The target substance detection chip according to any one of <1> to <5>, wherein a side wall portion is disposed.
<7> The target substance detection chip according to any one of <1> to <6>, a power supply unit connected to the electrode using an SPR excitation layer as an electrode, and total reflection from the back side of the target substance detection chip A light irradiator that can irradiate light under certain conditions, and a target light that is arranged on the surface side of the target substance detection chip and is generated due to the presence of the target substance in the liquid sample introduced to the surface side A target substance detection device comprising: a photodetection unit capable of being detected.
<8> The target substance detection device according to <7>, wherein the light irradiation unit can emit non-polarized light.
<9> A liquid sample introduction step of introducing a liquid sample onto the surface of the target substance detection chip according to any one of <1> to <6>, and the electrode and the exposed waveguide using the SPR excitation layer as an electrode. An electric field generating step for generating an electric field that draws the target substance in the liquid sample toward any one of the mode excitation layers; a light irradiation step for irradiating light under a total reflection condition from the back side of the target substance detection chip; and A target substance detection comprising: a light detection step of detecting, from the surface side of the target substance detection chip, light to be detected caused by the presence of the target substance in the liquid sample based on a light irradiation step Method.
<10> The target substance detection method according to <9>, wherein the light irradiation step is a step of irradiating non-polarized light.

  According to the present invention, the above-mentioned problems in the prior art can be solved, the target substance detection chip capable of detecting the target substance with high sensitivity, not only on the electrode but also the periphery thereof, and the target substance A detection apparatus and a target substance detection method can be provided.

It is explanatory 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 schematic structure of the target substance detection apparatus which concerns on one Embodiment of this invention. It is a figure which shows the mode of the detection area | region at the time of irradiating the s-polarized light in the target substance detection apparatus which concerns on an Example. It is a figure which shows the mode of the detection area | region at the time of irradiating the p-polarized light in the target substance detection apparatus based on an Example. In the target substance detection apparatus which concerns on an Example, it is a figure which shows the mode of the detection area at the time of irradiating non-polarized light.

(Target substance detection chip)
The target substance detection chip of the present invention includes a light-transmitting substrate, a waveguide mode excitation layer, and an SPR excitation layer, and irradiates the surface of the light-transmitting substrate side as a back surface under total reflection conditions from the back surface side. By the applied light, near-field light can be formed on the exposed surface of the waveguide mode excitation layer and the SPR excitation layer.
In the present specification, “near-field light” refers to light constituting the enhanced electric field. The near-field light is formed only near the surface of the target substance detection chip, and has a property of abruptly attenuating as the distance from the target substance detection chip increases.

<Light transmissive substrate>
The light transmissive substrate is a member that transmits light.
There is no restriction | limiting in particular as said light transmissive substrate, According to the objective, it can select suitably, For example, well-known light transmissive substrates, such as a glass substrate and a plastic substrate, can be used.
In the present specification, “light transmissivity” indicates that the visible light transmittance is 0.5% or more.

<Waveguide mode excitation layer>
The waveguide mode excitation layer is a layer in which a first layer and a second layer are laminated in this order on the light transmissive substrate, and the waveguide mode can be excited.
In the waveguide mode excitation layer, the waveguide mode is excited in the second layer on the surface side by the light irradiated under the total reflection condition from the back surface side of the target substance detection chip, and the enhancement is performed on the surface. An electric field is formed.

The first layer is formed of a metal material, a semiconductor material, and a first dielectric material.
There is no restriction | limiting in particular as said metal material, For example, metal materials, such as gold | metal | money, silver, copper, are mentioned.
There is no restriction | limiting in particular as said semiconductor material, Although well-known semiconductor materials or compound semiconductor materials, such as Si, Ge, SiGe, are mentioned, Among these, Si and Ge which are easy to obtain a high refractive index of 3.0 or more are preferable. .
As the first dielectric material is not particularly limited, for example, TiO 2, _ITO, although known optical transparent dielectric material such as ZnO may be mentioned, among others, 3.0 higher than the refractive index TiO ₂ obtained and having a low extinction coefficient (high light transmittance) is preferable.
In the present specification, the “refractive index” indicates a refractive index at a wavelength of light used for exciting the waveguide mode.
The thickness of the first layer is determined to be an optimum value depending on the constituent material and the wavelength of light to be irradiated, and this value is known to be calculated from the calculation using the Fresnel equation. Yes. Generally, when using light in a wavelength band from the near ultraviolet to the near infrared region, the first thickness is several nm to several hundred nm.

The second layer is formed of a second dielectric material.
The second dielectric material is not particularly limited. For example, a resin material such as silicone and PMMA (Polymethyl methacrylate), a glass material such as BK7 glass, an oxide such as SiO ₂ and TiO ₂ , and a nitride such as AlN And fluorides such as MgF ₂ , CaF, etc. Among them, SiO ₂ , PMMA, BK7 glass, which has a low refractive index of 1.35 to 1.5 and a low extinction coefficient, can be obtained. preferable. The second layer may be formed by laminating a plurality of types of the second dielectric material.
In addition, the thickness of the second layer is determined by the constituent material and the wavelength of light to be irradiated in the same manner as the first layer, and this value is calculated from the calculation using the Fresnel equation. It is known to be possible. In general, the thickness of the second layer is several tens of nm to several μm.
The formation method of the first layer and the second layer can be appropriately selected from known formation methods depending on the material.

<SPR excitation layer>
The SPR excitation layer is configured to be laminated on the waveguide mode excitation layer in a state where a part of the waveguide mode excitation layer is exposed, and the SPR can be excited.
The SPR excitation layer is made of a metal material, and acts as an electrode in addition to the excitation of the SPR. That is, the SPR excitation layer functions as the electrode that attracts the target substance by electrophoresis or dielectrophoresis, and makes it possible to detect the attracted target substance with high sensitivity using the excitation of the SPR.
On the other hand, the waveguide mode excitation layer that is partially exposed exists around the SPR excitation layer, and the target substance that is not attracted to the electrode is also introduced by the waveguide mode excitation layer. High sensitivity detection using wave mode excitation becomes possible.
The SPR excitation layer can also act as an electrode that keeps the target substance away according to the setting of electrophoresis or dielectrophoresis. In this case, the target substance moved away is concentrated on the exposed waveguide mode excitation layer and can be detected with high sensitivity by excitation of the waveguide mode.
On the other hand, even for the target substance remaining on the SPR excitation layer that cannot be moved away toward the waveguide mode excitation layer, the SPR excitation layer can be highly sensitively detected using the excitation of the SPR. The
Furthermore, it is possible to observe the movement of the target substance on the waveguide mode excitation layer and the SPR excitation layer by the electrophoresis or the dielectrophoresis using the SPR excitation layer as the electrode. Become.
Examples of the metal material constituting the SPR excitation layer include metal materials such as Au, Ag, Pt, and Al. Among these, Au, Ag, and Al are preferable, and these are the target substance detection chip. Is selected according to the wavelength of the light applied to the substrate, Au is preferable when the wavelength is 600 nm to 1,700 nm, Ag is preferable when the wavelength is 550 nm to 1,700 nm, and Al is preferable when the wavelength is 250 nm to 550 nm. .
As for the thickness of the SPR excitation layer, the optimum value is determined by the constituent material and the wavelength of the irradiated light, as in the waveguide mode excitation layer, but this value can be calculated from the calculation using the Fresnel equation. It is known that there is. In general, when the SPR is excited in the near ultraviolet to near infrared region, the thickness of the SPR excitation layer is several nm to several tens of nm.

There is no restriction | limiting in particular as a formation method of the said SPR excitation layer, Well-known formation methods, such as a vapor deposition method, sputtering method, CVD method, PVD method, a spin coat method, are mentioned.
The shape of the SPR excitation layer is not limited as long as it is a structure laminated on the waveguide mode excitation layer, and may be processed into an arbitrary shape from the viewpoint of suitably acting as the electrode. .

When observing light emission from the target substance or a labeling substance that labels the target substance, the target substance and the labeling substance were obtained from the SPR when the target substance and the labeling substance were close to the SPR excitation layer. There is a case where a phenomenon called quenching occurs in which energy is transferred to the metal layer and luminous efficiency is lowered.
In this case, when the coating layer is formed on the surface of the SPR excitation layer for the purpose of separating the target substance and the labeling substance from the surface of the SPR excitation layer, the quenching is suppressed and the decrease in luminous efficiency is suppressed. can do.
There is no restriction | limiting in particular as said coating layer, The layer with a thickness of several nanometers-tens of nanometers formed with transparent materials, such as aluminum oxide, glass materials, such as a silica glass, an organic polymer material, etc. is mentioned.

The number of SPR excitation layers formed is not particularly limited, and one SPR excitation layer may be provided on a part of the surface of the waveguide mode excitation layer as an electrode for attracting the target substance. In the known electrophoresis and dielectrophoresis, two electrodes are used as an electrode pair to generate an electric field that attracts the target substance to one electrode. When the number of SPR excitation layers is one, the electrode pair As the remaining one electrode constituting the electrode, the electrode outside the target substance detection chip may be used.
However, if the number of the SPR excitation layers formed is two or more, the SPR excitation layers can constitute the electrode pairs, and it is easier to further reduce detection leakage of the target substance between the electrode pairs. can do.

In the target substance detection chip, fluorescence observation of the target substance or the like is suitably performed by setting the wavelength of the near-field light generated on the surface of the target substance detection chip according to the target substance (or the labeling substance). be able to. That is, when the wavelength of the near-field light belongs to the wavelength band of the excitation wavelength of the fluorescence of the target substance or the like, the fluorescence observation of the target substance accompanying the excitation of the target substance or the like can be suitably performed.
In the target substance detection chip, since both the SPR excitation layer and the waveguide mode excitation layer serve as means for detecting the target substance by forming the near-field light, the SPR excitation layer and the waveguide mode excitation are used. One ideal aspect is that the wavelength of the near-field light generated on each surface of the layer is equal to the fluorescence excitation wavelength of the target substance or the labeling substance to be detected.
Therefore, it is preferable that the difference in the peak position of the wavelength of the near-field light generated on the surfaces of the waveguide mode excitation layer and the SPR excitation layer is at most 40 nm.

The SPR excitation layer cannot excite the SPR with s-polarized light. On the other hand, the waveguide mode excitation layer can excite either the s-polarized light or the p-polarized light, but the s-polarized light can excite the waveguide mode with higher efficiency.
Therefore, when the s-polarized light is used, the SPR cannot be excited by the SPR excitation layer, and when the p-polarized light is used, it is difficult to excite the waveguide mode excitation layer with high efficiency.
On the other hand, if non-polarized light is used, both the SPR excitation layer and the waveguide mode excitation layer can be effectively excited. As a result, the SPR excitation layer and the waveguide mode excitation layer The near-field light can be formed on both surfaces.

<Design method>
In the detection chip using the excitation of the SPR, a metal thin film of a metal material that excites the SPR has been formed on a glass substrate so far (see, for example, Patent Document 2).
Therefore, the target substance detection chip in which the SPR excitation layer is formed on the waveguide mode excitation layer is a detection chip having a novel layer structure.
Therefore, it is a problem whether the SPR excitation layer formed on the waveguide mode excitation layer forms the near-field light. However, this is a case where the SPR excitation layer is formed on the waveguide mode excitation layer. Even so, it was confirmed that the SPR excitation layer can form the near-field light (see Examples below). The optical characteristics of the SPR excitation layer formed on the waveguide mode excitation layer are confirmed to follow the optical characteristics set by a setting method based on the existing Fresnel equation. As the SPR excitation layer, The SPR excitation layer can be designed by an existing setting method.
Hereinafter, a configuration example of the target substance detection chip having the waveguide mode excitation layer in addition to the SPR excitation layer will be specifically described from the viewpoint of a design method.

First, the target substance or the labeling substance to be detected is selected, and the excitation wavelength of these fluorescences is determined.
Next, a material for forming the SPR excitation layer that generates the near-field light having a wavelength belonging to the wavelength band of the excitation wavelength is selected, and an incident angle of light suitable for excitation of the SPR by a setting method based on the Fresnel equation And determining the thickness of the SPR excitation layer. Thereby, a suitable SPR excitation layer is set.
Next, the first waveguide mode excitation layer is configured by the setting method based on the Fresnel equation so that the waveguide mode is excited based on the excitation wavelength and the incident angle of the light specified above. The forming material and thickness of each of the layers and the second layer are determined. Thereby, a suitable waveguide mode excitation layer is set.
Here, the setting of the SPR excitation layer is performed prior to the setting of the waveguide mode excitation layer because the SPR excitation layer has a design range of the wavelength of the near-field light rather than the waveguide mode excitation layer. If the wavelength of the near-field light that can be set for the SPR excitation layer is narrow, the setting condition can be satisfied for the waveguide mode excitation layer.
Finally, the thickness of the SPR excitation layer is finely adjusted so that the SPR is excited most efficiently with respect to the structure in which the SPR excitation layer is formed on the waveguide mode excitation layer.
Note that while the SPR excitation layer cannot excite the SPR with the s-polarized light, the waveguide mode excitation layer can be excited with either the p-polarized light or the s-polarized light. The s-polarized light can excite the waveguide mode with high efficiency.
From this, when calculating conditions suitable for excitation of the SPR excitation layer, calculation is performed using the p-polarized light, and when calculating conditions suitable for excitation of the waveguide mode excitation layer, Calculation is performed using the s-polarized light. When the target substance is detected using the actually manufactured target substance detection chip, excitation light including both the p-polarized light and the s-polarized light is used to simultaneously excite the SPR and the waveguide mode. Irradiation is preferred.
As the light including both the p-polarized light and the s-polarized light, it is easiest to use the non-polarized light, and it is suitable for simultaneous excitation of the SPR excitation layer and the waveguide mode excitation layer. .

As a specific example, a case where beads Bright Blue (Fluoresbrite (registered trademark), Polyscience) that emits fluorescence having a wavelength of 407 nm and light having an excitation wavelength of 375 nm is detected as the target substance will be described.
First, Al is selected as a material for forming the SPR excitation layer that forms the near-field light having the excitation wavelength of 375 nm. Further, the wavelength of incident light incident on the SPR excitation layer to obtain the near-field light is equal to the excitation wavelength and is 375 nm.
The optimum incident angle for exciting the SPR with respect to the Al layer on the silica glass substrate by the p-polarized light having a wavelength of 375 nm and the thickness of the Al layer are set to 72 according to the setting method based on the Fresnel equation, respectively. .3 ° and 16 nm.
Next, the layer structure of the waveguide mode excitation layer capable of exciting the waveguide mode at an incident angle of 72.3 ° with s-polarized light having a wavelength of 375 nm is determined. Here, as an example, a case is assumed in which a TiO ₂ layer as the _first layer and a SiO ₂ layer as the _second layer are laminated in this order on a silica glass substrate.
When the thickness of the TiO ₂ layer and the SiO ₂ layer is optimized by the setting method based on the Fresnel equation so that the guided mode is excited by the s-polarized light and the incident angle thereof, the optimum The thickness is determined to be 39.2 nm and 241 nm, respectively.
Furthermore, in the case where the Al layer is laminated on the waveguide mode excitation layer composed of the TiO ₂ layer having a thickness of 39.2 nm and the SiO ₂ layer having a thickness of 241 nm, p-polarized light having a wavelength of 375 nm is used. The thickness of the Al layer as the SPR excitation layer is readjusted by a setting method based on the Fresnel equation so that SPR is excited most strongly. Then, the optimum thickness of the Al layer is determined to be 12 nm.
In this way, the incident wavelength, incident angle, and layer structure are determined.
Based on the above settings, the target substance detection chip actually manufactured may be irradiated with non-polarized light having a wavelength of 375 nm at an incident angle of 72.3 °. Thereby, in the target substance detection chip, the SPR and the waveguide mode can be excited simultaneously.

As described above, the material for forming the SPR excitation layer is preferably Au when the wavelength of the incident light is 600 nm to 1,700 nm, Ag is preferably 550 nm to 1,700 nm, and Al is 250 nm to 550 nm. preferable.
Therefore, as a combination of the SPR excitation layer and the waveguide mode excitation layer, for example, when ultraviolet light is used as the incident light, the SPR excitation layer is formed of Al, and the first in the waveguide mode excitation layer. forming a second layer with SiO _2, the first layer is mentioned to be formed by TiO _2, when using the visible light to the incident light, the SPR excitation layer is formed of Au or Ag, the conductive For example, the second layer of the wave mode excitation layer may be formed of SiO ₂ and the first layer may be formed of TiO ₂ or Si.
In addition, as a design example when the excitation wavelength of the target substance or the like is viewed from (1) when the excitation wavelength is 375 nm, the incident angle is 72.3 °, and the SPR excitation layer has a thickness of 12 nm. Forming an Al layer, forming the second layer of the waveguide mode excitation layer as a SiO ₂ layer having a thickness of 241 nm, and forming the first layer as a TiO ₂ layer having a thickness of 39.2 nm. (2) When the excitation wavelength is 700 nm, the incident angle is set to 72.3 °, the SPR excitation layer is formed of an Au layer having a thickness of 25 nm, and the second in the waveguide mode excitation layer layer thickness is formed in the SiO ₂ layer of 495nm in thickness and wherein the first layer include forming at TiO ₂ layer of 89.2nm, (3) when the excitation wavelength is 500 nm, the Angle of incidence As 69.8 °, the thickness of the SPR excitation layer is made of Al layer of 14 nm, a thickness of the second layer in the waveguide mode excitation layer is formed of SiO ₂ layer of 284 nm, the first For example, the layer may be a TiO ₂ layer having a thickness of 60.4 nm.
In the target substance detection chip according to the design example (1), the electric field strength N at the outermost surface of the waveguide mode excitation layer is estimated to be 42.2, and the electric field strength at the outermost surface of the SPR excitation layer is estimated. N is estimated to be 14.9. Here, the numerical value of the electric field strength N is a relative value when the electric field strength of incident light is 1.

<Others>
When the target substance detection chip is formed in a plate shape in which the front surface and the back surface are parallel, the light irradiated from the back surface side is not totally reflected when a liquid exists 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 the light at a specific angle. And is introduced into the target substance detection chip, and the light introduced into the target substance detection chip is irradiated on the surface under the total reflection condition to form near-field light in the vicinity of the surface. A target substance detection chip may be configured. Alternatively, the front surface and the back surface may be formed so as not to be parallel. Alternatively, the back surface of the target substance detection chip may be irradiated with the light via a known optical prism.
The optical prism can be optically bonded to the back surface of the target substance detection chip using a refractive index adjusting oil or an optical adhesive. Further, when the same material as the material for forming the light transmissive substrate is selected as the material for forming the optical prism, a material in which the light transmissive substrate and the optical prism are integrally molded may be used. it can.

In addition, a liquid sample to be detected by the target substance is introduced onto the surface of the target substance detection chip. As a method for holding the liquid sample on the surface of the target substance detection chip, for example, the liquid sample is dropped on the surface of the target substance detection chip and then covered with a cover glass or the like.
Further, in order to securely hold the liquid sample, a liquid sample tank may be formed on the surface of the target substance detection chip. Although there is no restriction | limiting in particular as said liquid sample tank, From the viewpoint of setting it as a simple structure, on the surface of these layers so that the whole said waveguide mode excitation layer and said SPR excitation layer which are exposed may be enclosed. It is preferable to be configured by being provided with a side wall portion that is erected on the surface and serves as a component portion of the liquid sample tank with the surface as a bottom.
In addition, there is no restriction | limiting in particular as a formation material of the said side wall part, A well-known glass material, a resin material, etc. can be mentioned, As a formation method of the said side wall part, the well-known method according to material is mentioned. it can.

  Next, a configuration example of the target substance chip of the present invention will be specifically described with reference to FIG. FIG. 1 is an explanatory diagram showing a schematic configuration of a target substance detection chip according to an embodiment of the present invention.

As shown in FIG. 1, the target substance detection chip 1 includes a light transmissive substrate 2, a first layer 3 and a second layer 4 formed on the light transmissive substrate 2 in this order. A waveguide mode excitation layer 5 configured to be able to excite the waveguide mode, and laminated with the waveguide mode excitation layer 5 partially exposed on the waveguide mode excitation layer 5; SPR excitation layers 6a and 6b that can excite the SPR are arranged. For the purpose of exciting the waveguide mode, the first layer 3 is made of the metal material, the semiconductor material, and the first dielectric material, and the second layer 4 is made of the second dielectric material. Made of body material.
In the target substance detection chip 1, the surface of the waveguide mode excitation layer 5 and the SPR excitation layers 6a and 6b exposed by light irradiated from the rear surface side under the total reflection condition with the surface on the light transmissive substrate 2 side as the back surface. Near-field light can be formed on the top.

The two SPR excitation layers 6a and 6b are arranged as two or more layers separated from each other, and can constitute the electrode pair.
Further, in the target substance detection chip 1, the waveguide mode excitation layer 5 and the SPR excitation layers 6a and 6b that are exposed are erected on the surfaces of these layers so as to surround all or part of the exposed waveguide mode excitation layer 5 and the SPR excitation layers 6a and 6b. A side wall 7 serving as a component of the liquid sample tank is disposed, and the liquid sample is introduced and held in the liquid sample tank. Moreover, the area | region on the surface of the target substance detection chip 1 enclosed by the side wall part 7 is made into a detection area, and the said light is irradiated from the back surface side of the target substance detection chip 1 corresponding to this position.

  In the target substance detection chip 1 configured as described above, when a voltage is applied to the SPR excitation layers 6a and 6b as the anode / cathode electrode pair, one of the SPR excitation layers 6a and 6b is selected depending on the polarity. The target substance is attracted to a position on the surface, and highly sensitive detection in which the target substance is concentrated at the position can be performed. Further, the target substance positioned on the surface of the waveguide mode excitation layer 5 without being attracted on the surface of any of the SPR excitation layers 6a and 6b can be detected by the waveguide mode excitation layer 5. It is possible to perform detection with high sensitivity by suppressing detection omission.

(Target substance detection device)
The target substance detection device of the present invention is configured by arranging the target substance detection chip, power supply unit, light irradiation unit, and light detection unit of the present invention.
In the target substance detection device, the target substance or the labeling substance that labels the target substance is fluorescently emitted by the near-field light accompanying the excitation of the SPR and the waveguide mode in the target substance detection chip, or Scattered light is generated, and the target substance is detected using the fluorescence and / or scattered light as light to be detected.
The labeling substance is not particularly limited, and examples thereof include fluorescent labeling substances and light scattering substances that 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, and fluorescent dyes 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 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 bond, chelate bond, amino bond, and the like can be used. Can be applied.

<Power supply unit>
The power supply unit is a unit connected to the electrode using the SPR excitation layer as an electrode.
There is no restriction | limiting in particular as said power supply part, The power supply part used for a well-known electrophoresis apparatus and a dielectrophoresis apparatus is mentioned.

<Light irradiation part>
The light irradiating unit is a unit that can irradiate light from the back side of the target substance detection chip under total reflection conditions.
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 | ramp, LED, a laser, etc. are mentioned.
In the present invention, the near-field light is formed on the surface of the target substance detection chip by irradiating light from the back side of the target substance detection chip under total reflection conditions, and the target substance based on the near-field light, etc. The detected light from is generated.
Therefore, the role required for the light irradiation part is only to irradiate light from the back side of the target substance detection chip under the total reflection condition. If it plays such a role, it is limited to the selection of the light source. There is no.

In the case of using a radiation light source such as a lamp or LED, in order to avoid leakage of irradiation light from the front surface side of the target substance detection chip, the back surface side of the target substance detection chip is irradiated out of the emitted light. Light in all directions must satisfy the total reflection condition. For this reason, when a radiation light source is used, a guide unit such as a collimator lens that restricts the irradiation direction of irradiation light to a specific direction may be used.
When the detection light is fluorescence, a monochromatic light source having a wavelength capable of exciting the fluorescence is used, or light from a light source having a wide wavelength band such as a lamp or LED is used as an optical filter such as a bandpass filter. It is preferable to irradiate from the back side of the target substance detection chip after extracting only a wavelength that can be transmitted and monochromatic and excite fluorescence.

Moreover, it is preferable that it is the said non-polarized light as the light irradiated on the total reflection conditions from the back surface side of the said target substance detection chip | tip by the said light irradiation part.
When the light is non-polarized light, the SPR excitation layer and the waveguide mode excitation layer can be most easily simultaneously excited as light including both the s-polarized light and the p-polarized light.

<Light detector>
The light detection unit is a unit that is disposed on the surface side of the target substance detection chip, and is capable of detecting light to be detected generated due to the presence of the target substance in the liquid sample introduced to the surface side. .
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 the information of the optical signal can be acquired as two-dimensional image information, the position information of the optical signal in the two-dimensional image information that appears as a light spot, the size information grasped from the two-dimensional image, the optical signal at the light spot By observing the intensity increase / decrease information in time series, it is possible to obtain information on whether the light spot is due to the target substance or the like. Furthermore, when the two-dimensional image is acquired as a moving image, it is possible to easily observe how the optical signal moves between the electrodes.
In order to obtain such two-dimensional image information, an imaging device may be selected as the light detection unit. There is no restriction | limiting in particular as said imaging device, According to the objective, it can select suitably, Image sensors, such as a well-known CCD image sensor and a CMOS image sensor, can be used.

(Target substance detection method)
The target substance detection method of the present invention relates to a target substance detection method using the target substance detection chip of the present invention, and includes a liquid sample introduction step, an electric field generation step, a light irradiation step, and a light detection step.

<Liquid sample introduction process>
The liquid sample introduction step is a step of introducing the liquid sample onto the surface of the target substance detection chip.
The method for performing the liquid sample process is not particularly limited, and the liquid sample dropped on the surface of the target substance detection chip is covered with a cover glass and held, or the liquid sample is placed on the target substance detection chip. When a tank is formed, the liquid sample is introduced into the liquid sample tank.

<Electric field generation process>
The electric field generating step is a step of generating an electric field that draws the target substance in the liquid sample toward either the electrode or the exposed waveguide mode excitation layer using the SPR excitation layer as an electrode.
There is no restriction | limiting in particular as the implementation method of the said electric field generation process, Applying a voltage to the said SPR excitation layer using the said power supply part in the said target substance detection apparatus is mentioned. Note that whether the target substance is attracted to the electrode (the SPR excitation layer) or the waveguide mode excitation layer depends on the setting of the power supply unit, and an electric field that imparts the action of attracting the objective substance to the electrode. Or by generating an electric field that imparts an action of moving the target substance away from the electrode.

<Light irradiation process>
The said light irradiation process is a process of irradiating light on the total reflection conditions from the back surface side of the said target substance detection chip.
There is no restriction | limiting in particular as the implementation method of the said light irradiation process, Irradiating the said light using the said light irradiation part in the said target substance detection apparatus is mentioned.

<Light detection process>
The light detection step is a step of detecting the detected light generated from the presence of the target substance in the liquid sample from the surface side of the target substance detection chip based on the light irradiation step.
There is no restriction | limiting in particular as the implementation method of the said light detection process, The detection of the said to-be-detected light using the said light detection part in the said target substance detection apparatus is mentioned.

  Next, an example of the target substance detection apparatus and the target substance detection method of the present invention will be specifically described with reference to FIG. FIG. 2 is an explanatory diagram showing a schematic configuration of a target substance detection apparatus according to an embodiment of the present invention.

As illustrated in FIG. 2, the target substance detection device 100 includes a target substance detection chip 1, an optical prism 8, a power supply unit (not shown), a light irradiation unit 101, and a light detection unit 102.
The configuration and effects of the target substance detection chip 1 are the same as those described with reference to FIG.
The optical prism 8 is disposed in optical contact with the back surface of the target substance detection chip 1.
The power supply unit is configured to be connected to the electrode using the SPR excitation layer as an electrode.
The light irradiation unit 101 is configured to be able to irradiate light from the back side of the target substance detection chip 1 under total reflection conditions.
The light detection unit 102 is arranged on the surface side of the target substance detection chip 1 and is configured to be able to detect the detection light generated due to the presence of the target substance in the liquid sample A introduced to the surface side.

As the target substance detection method using such a target substance detection apparatus 100, first, a liquid sample is introduced onto the surface of the target substance detection chip 1, that is, into the liquid sample tank surrounded by the side wall portion 7. Liquid sample A is introduced (liquid sample introduction step).
Next, the SPR excitation layers 6a and 6b are used as electrodes to generate an electric field that draws the target substance toward the electrodes, that is, a voltage is applied from the power supply unit to the electrode pairs of the SPR excitation layers 6a and 6b (electric field generation step). .
Next, the light irradiation unit 101 is used to irradiate light from the back surface side of the target substance detection chip 1 under total reflection conditions (light irradiation process).
Finally, the detected light generated due to the presence of the target substance in the liquid sample A is detected from the surface side of the target substance detection chip 1 by the light detection unit 102 (light detection step).
The electric field generation step, the light irradiation step, and the light detection step may be performed in the order of performing the electric field generation step while performing the light irradiation step and the light detection step. When implemented in this order, it is possible to detect the movement of the target substance by the electric field.

In the target substance detection apparatus 100 and the target substance detection method configured as described above, the target substance is attracted to a position on the surface of one of the SPR excitation layers 6a and 6b by voltage application from the power supply unit, Sensitive detection in which the target substance is concentrated at the position can be performed. Further, the target substance positioned on the surface of the waveguide mode excitation layer 5 without being attracted on the surface of any of the SPR excitation layers 6a and 6b can be detected by the waveguide mode excitation layer 5. It is possible to perform detection with high sensitivity by suppressing detection omission.
Moreover, when the light irradiated from the light irradiation unit 101 is the non-polarized light, the SPR excitation layers 6a and 6b and the waveguide mode excitation layer 5 can be excited simultaneously with high efficiency. Substances can be detected with higher sensitivity.
Further, when the optical signal detected by the light detection unit 102 is acquired as a two-dimensional image because the near-field light formed by the SPR excitation layers 6a and 6b and the waveguide mode excitation layer 5 is limited to the vicinity of the surface. While the background is a dark field, the detected light generated due to the presence of the target substance is displayed as a light spot, so that the target substance can be clearly detected.

According to the configuration of the target substance detection chip 1 shown in FIG. 1, the target substance detection chip according to the example was manufactured as follows.
First, a SiO ₂ substrate (manufactured by Eiko Co., Ltd.) having a thickness of 1 mm was prepared as the light transmissive substrate 2.
Next, the first layer 3 made of TiO ₂ having a thickness of 32 nm was formed on the light-transmitting substrate 2 by a radio frequency (RF) magnetron sputtering apparatus (manufactured by Shibaura Mechatronics Co., Ltd., CFS-4EP-L).
Next, a second layer 4 made of SiO ₂ having a thickness of 240 nm was formed on the first layer 3 by the radio frequency (RF) magnetron sputtering apparatus.
Next, Al SPR excitation layers 6a and 6b each having a thickness of 12 nm were formed on the second layer 4 by using the radio frequency (RF) magnetron sputtering apparatus.
On the SPR excitation layers 6a and 6b, an Al natural oxide film of about 4 nm is formed by oxygen in the air.
In the target substance detection chip according to the example, the side wall portion 7 is not formed.

While using the target substance detection chip according to the example manufactured as described above, the target substance detection apparatus according to the example was manufactured as follows according to the configuration of the target substance detection apparatus 100 shown in FIG.
First, the target substance detection chip according to the example was placed on a trapezoidal prism having an inclination angle of 42 ° (the inclination angle corresponds to the angle indicated by φ in FIG. 2).
Next, using an LED light source (M375F2 manufactured by Thorlabs, Inc.) having a wavelength of irradiation light of 375 nm, light can be introduced into the optical prism 8 by an optical fiber having a core diameter of 600 μm with a collimating lens attached to the emission end. The light irradiation unit 101 is set so that any one of s-polarized light, p-polarized light, and non-polarized light can be selected and incident on the target substance detection chip 1 by a polarizing filter installed between the end and the optical prism 8. did.
Here, the incident angle θ (see θ in FIG. 2) of the light applied to the back surface of the target substance detection chip 1 was set to 72.3 °.
Finally, an optical microscope provided with a 10 × objective lens and a cooled CMOS camera as the light detection unit 102 was disposed above the surface side of the target substance detection chip 1. Note that the shutter time of the camera is 100 ms.

Using the target substance detection apparatus according to the example manufactured as described above, a target substance detection test was performed as follows.
First, as the target substance, fluorescent beads Bright Blue (Fluoresbrite (registered trademark), Polyscience) that emits fluorescence having an excitation wavelength of 375 nm and an excitation wavelength of 407 nm were used.
Subsequently, the fluorescent beads were added to water, and a liquid sample A was prepared in such a manner that the fluorescent beads were diluted 1,000 times in volume ratio with water.
Next, the liquid sample A was dropped on the surface of the target substance detection chip 1 in an amount of about 5 μL. Further, the state in which the dropped liquid sample A covers the surface of the SPR excitation layers 6a and 6b and the exposed waveguide mode excitation layer 5 was held by the cover glass.
Finally, the state on the surface of the target substance detection chip 1 irradiated with light from the light irradiation unit 101 was photographed by the light detection unit 102 to detect the target substance.

The target substance was detected three times by switching the irradiation light of the light irradiation unit 101 to s-polarized light, p-polarized light, and non-polarized light. The intensity of light when the irradiation light is s-polarized light is 225 μW, the intensity of light when the irradiation light is p-polarization is 147 μW, and the light intensity when the irradiation light is non-polarized light The intensity was 958 μW.
2D images taken by the light detection unit 102 are shown in FIGS. FIG. 3 is a diagram illustrating a state of a detection region when s-polarized light is irradiated in the target substance detection apparatus according to the embodiment. FIG. 4 illustrates p-polarization in the target substance detection apparatus according to the embodiment. FIG. 5 is a diagram illustrating a state of the detection region when non-polarized light is irradiated in the target substance detection device according to the example.

As shown in FIG. 3, in the s-polarized light, it is difficult to catch the detected light from the target substance in the detection region on the SPR excitation layer 6a (and the SPR excitation layer 6b), while the guided mode excitation layer 5 The detected light from the target substance can be clearly captured in the upper detection region.
As shown in FIG. 4, the p-polarized light can clearly detect the detected light from the target substance in the detection region on the SPR excitation layer 6a (and the SPR excitation layer 6b). In the detection region on the wave mode excitation layer 5, the detected light from the target substance is somewhat unclear compared to FIG. 3 (s-polarized light).
As shown in FIG. 5, in the non-polarized light, in the detection region of both the detection region on the SPR excitation layer 6 a (and the SPR excitation layer 6 b) and the detection region on the waveguide mode excitation layer 5, The detected light can be clearly captured. Note that the fluorescence intensity of the detected light in the detection region on the SPR excitation layer 6a (and the SPR excitation layer 6b) averages 10,010 [a. u. The average fluorescence intensity of the detected light in the detection region on the waveguide mode excitation layer 5 is 14,585 [a. u. ]Met.
Further, the detected light in both detection regions shown in FIG. 5 (non-polarized light) was the strongest light compared to those in FIG. 3 (s-polarized light) and FIG. 4 (p-polarized light).

DESCRIPTION OF SYMBOLS 1 Target substance detection chip 2 Light transmissive substrate 3 1st layer 4 2nd layer 5 Waveguide mode excitation layer 6a, 6b SPR excitation layer (electrode)
7 Side wall part 8 Optical prism 100 Target substance detection apparatus 101 Light irradiation part 102 Light detection part A Liquid sample θ Incident angle φ Inclination angle

Claims (10)

A light transmissive substrate;
A first layer formed of any one of a metal material, a semiconductor material, and a first dielectric material and a second layer formed of a second dielectric material on the light-transmitting substrate in this order. A waveguide mode excitation layer configured to be laminated and capable of exciting the waveguide mode;
An SPR excitation layer that is configured to be laminated on the waveguide mode excitation layer in a state in which a part of the waveguide mode excitation layer is exposed, and an SPR excitation layer capable of exciting SPR is disposed,
Near-field light can be formed on the exposed surface of the waveguide mode excitation layer and the SPR excitation layer by light irradiated from the back surface side under the total reflection condition with the light transmitting substrate side surface as the back surface. A target substance detection chip.   The target substance detection chip according to claim 1, wherein the SPR excitation layers are arranged as two or more layers separated from each other.   3. The target substance detection chip according to claim 1, wherein the difference between the peak positions of the wavelengths of near-field light formed on the surfaces of the waveguide mode excitation layer and the SPR excitation layer is at most 40 nm.   The target substance detection chip according to any one of claims 1 to 3, wherein a material for forming the SPR excitation layer is Au, Ag, or Al. The material for forming the first layer is any one of Si, Ge, SiGe, and TiO ₂ , and the material for forming the second layer is any one of SiO ₂ , PMMA, and BK7 glass. The target substance detection chip according to claim 1.   Further, a side wall is provided standing on the surface of the waveguide mode excitation layer and the SPR excitation layer to be exposed or part of the layer, and serves as a component of the liquid sample tank with the surface as a bottom. The target substance detection chip according to any one of claims 1 to 5, wherein a portion is arranged. The target substance detection chip according to any one of claims 1 to 6,
A power supply connected to the electrode using the SPR excitation layer as an electrode;
A light irradiator capable of irradiating light from the back side of the target substance detection chip under total reflection conditions;
A light detection unit arranged on the surface side of the target substance detection chip and capable of detecting detected light caused by the presence of the target substance in the liquid sample introduced to the surface side;
The target substance detection apparatus characterized by having.   The target substance detection device according to claim 7, wherein the light irradiation unit can emit non-polarized light. A liquid sample introduction step of introducing a liquid sample onto the surface of the target substance detection chip according to claim 1;
An electric field generating step of generating an electric field that draws the target substance in the liquid sample toward either the electrode or the exposed waveguide mode excitation layer using the SPR excitation layer as an electrode;
A light irradiation step of irradiating light from the back side of the target substance detection chip under total reflection conditions;
A light detection step of detecting, from the surface side of the target substance detection chip, detected light caused by the presence of the target substance in the liquid sample based on the light irradiation step;
A target substance detection method comprising: The target substance detection method according to claim 9, wherein the light irradiation step is a step of irradiating non-polarized light.