JP5923811B2 - Target substance detection plate, target substance detection apparatus, and target substance detection method - Google Patents

Description

  The present invention relates to a target substance detection plate that detects a target substance contained in a sample liquid using an optical waveguide mode or surface plasmon resonance, a target substance detection apparatus having the target substance detection plate, and the target substance detection apparatus The present invention relates to a target substance detection method using the

  In recent years, there is a need for a portable, easy-to-operate and high-sensitivity detector in various fields such as medical examinations, pharmaceuticals, early detection of diseases and infectious diseases, environmental pollution detection, and anti-terrorism measures. As a sensor that is small enough to be carried and can measure various substances contained in a liquid, an SPR sensor using surface plasmon resonance (SPR) and an optical waveguide sensor using a waveguide mode are known. (For example, refer nonpatent literatures 1-19 and patent documents 1-7.). These sensors detect various biomarkers and viruses resulting from disease, selectively detect various biomaterials such as proteins, assess environmental pollution, detect toxic and illegal drugs used in terrorism, and explosives Has been used.

  FIG. 1 shows a configuration example of the most popular SPR sensor 200 called a Kretschmann arrangement. In this SPR sensor 200, a metal thin film layer 202 is formed by vapor deposition of a metal such as gold, silver, or aluminum on a transparent substrate 201, and an optical surface is formed on the surface of the transparent substrate 201 opposite to the surface on which the metal thin film layer 202 is formed. The prism 203 has a structure in which the prism 203 is in close contact, and the laser light emitted from the light source 204 is polarized by the polarizing plate 205 and irradiated to the transparent substrate 201 through the optical prism 203. Incident light 210A is incident under conditions of total reflection. Surface plasmon resonance appears at a certain incident angle by the evanescent wave that oozes out to the metal surface side of the incident light 210A. The incident angle θ is appropriately changed by driving the optical system. When surface plasmon resonance occurs, the evanescent wave is absorbed by the surface plasmon, so that the intensity of the reflected light is remarkably reduced near this incident angle. The condition for surface plasmon resonance to appear varies depending on the dielectric constant in the vicinity of the surface of the metal thin film layer 202. Therefore, if adsorption, approach, separation, or alteration of a substance occurs on the surface of the metal thin film layer 202, the intensity of the reflected light 210B is increased. Change appears. Therefore, when the detected sample is bonded to or adsorbed on the surface of the metal thin film layer 202 and the dielectric constant changes, the reflection characteristic of the incident light 210A changes, so that the reflected light reflected from the metal thin film layer 202 is reflected. The sample to be detected can be detected by monitoring the intensity change of 210B by the photodetector 206.

  The optical waveguide mode sensor has a structure very similar to that of the SPR sensor, and also detects a substance adsorption or a change in dielectric constant on the detection surface of the sensor. It is known that this optical waveguide mode sensor can use an optical system equivalent to all optical systems that can be used in the SPR sensor.

  FIG. 2 shows an optical waveguide mode sensor 400 using an arrangement similar to the Kretschmann arrangement. The optical waveguide mode sensor 400 includes a transparent substrate 401a, a thin film layer 401b composed of a metal layer or a semiconductor layer coated thereon, and an optical waveguide layer 401c formed on the thin film layer 401b. 401 is used. The optical prism 402 is brought into close contact with the surface of the detection plate 401 opposite to the surface on which the optical waveguide layer 401c is formed via refractive index adjusting oil. Incident light 410 </ b> A emitted from the light source 403 and polarized by the polarizing plate 404 is applied to the detection plate 401 through the optical prism 402. The incident light 410A is incident on the detection plate 401 under conditions that cause total reflection. When the incident light 410A is coupled with an optical waveguide mode (also called a leakage mode or leaky mode) propagating in the optical waveguide at a certain incident angle θ, the optical waveguide mode is excited, and light is reflected near the incident angle. The light intensity changes greatly. Such an optical waveguide mode excitation condition changes depending on the dielectric constant in the vicinity of the surface of the optical waveguide layer 401c. Therefore, if adsorption, approach, separation, or alteration of a substance occurs on the surface of the optical waveguide layer 401c, the reflected light 410B Changes appear in intensity. By observing this change with the photodetector 405, it is possible to detect phenomena such as adsorption, approach, separation, and alteration of substances on the surface of the optical waveguide layer 401c.

  An SPR sensor or an optical waveguide mode sensor has an effect of enhancing the light emission of a fluorescent material when a substance capable of light excitation light emission such as a fluorescent dye (hereinafter referred to as a fluorescent material) is attached to or brought close to the detection surface. . This effect is often used to amplify signals during substance detection. For example, when a desired specific substance is captured in the vicinity of the surface of the metal thin film layer 202 in FIG. 1, the specific substance is very small, the amount is remarkably small, or the dielectric constant is almost the same as that of the surrounding medium. In some cases, the method of observing the change in reflected light characteristics shown in FIG. 1 may not provide a sufficient signal. At this time, a fluorescent substance is attached to the captured specific substance and used as a label. The attached fluorescent substance is enhanced in light emission intensity due to the electric field enhancement effect of plasmons excited by excitation light, so that the capture of the specific substance can be indirectly detected with high sensitivity. This effect is also obtained in the vicinity of the surface of the optical waveguide layer 401c in FIG.

  Here, in both cases of FIGS. 1 and 2, light emission from the fluorescent material is mainly emitted to the side opposite to the side of the detection plate where the excitation light is irradiated, that is, the side where the fluorescent material is attached. Therefore, in order to detect this luminescence, a device for detecting the luminescence, for example, a photo detector such as a CCD, a photomultiplier tube, or a photodiode is connected to the detection surface side of the detection plate, that is, the side opposite to the surface on which the prism is arranged. Need to be placed in.

The SPR sensor and optical waveguide mode sensor shown above are already widely used and widely used. However, in the case of general measurement, the prism and detection as shown in FIG. 1 and FIG. In addition to the plate, when the target substance to be detected is transported to the surface of the detection surface, for example, when the subject is a liquid, the channel needs to be arranged on the surface of the detection plate. Therefore, there are problems that the number of parts increases and handling is not easy.
In actual use, it is necessary to join the prism, the detection plate, and the transportation path, but this joining process must be performed every time the detection plate or transportation path is replaced for each detection. There is a problem that the system becomes complicated.
In addition, the prism as a part generally requires high-precision polishing and is expensive.

An example in which the prism, the detection plate, and the transportation path are integrally formed is a biochip disclosed in Patent Document 7. In this biochip, a microfluidic channel as a transport path is formed on a substrate, and a plurality of wedge-shaped sharpened portions formed by first and second inclined surfaces are formed in the microfluidic channel. In addition, a metal layer that excites surface plasmons and a dielectric layer on which a capture molecule that specifically binds to a target molecule labeled with a phosphor is fixed are formed on the inclined surface of the sharpened portion. When the target substance is immobilized on the fluorescent material, fluorescence is detected from the phosphor excited through the surface plasmon.
According to this biochip, each part that functions as a prism, a detection plate, and a transport path is formed integrally with the substrate, and the number of parts can be reduced.
However, in this biochip, there is a problem in that the system is complicated and the production cost is high because the sharp part that forms the detection surface of the target molecule and the microfluidic channel as the transport path are formed separately. .
Moreover, establishment of an efficient detection method is demanded by arranging such a prism, a detection plate, and a transportation path on the plate.

Japanese Patent No. 4581135 Japanese Patent No. 4,595,072 JP 2007-271596 A JP 2008-46093 A JP 2009-85714 A International Publication No. 2010/87088 JP 2010-145408 A

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  An object of the present invention is to solve the above-described problems and achieve the following objects. That is, the present invention reduces the number of parts of the detection chip composed of the optical prism and the detection plate used in the SPR sensor and the optical waveguide mode sensor, can be easily manufactured in a small size and at a low cost, and the target substance can be obtained quickly and highly. An object of the present invention is to provide a target substance detection plate, a target substance detection device, and a target substance detection method that have a detection chip that can be detected with sensitivity and that allows easy introduction of an analyte liquid and that can efficiently measure the target substance. And

Means for solving the problems are as follows. That is,
<1> One or a plurality of concave accommodating portions for accommodating a detection chip for detecting a target substance, and a flow path for supplying a sample liquid for verifying the presence of the target substance to the accommodating portion are formed. A translucent plate main body and the detection chip housed in the housing portion, wherein the detection chip is disposed on one surface of the transparent base portion and a plate-like transparent base portion that transmits light And a detection groove that is connected to the flow path and into which the analyte liquid is introduced along the flow path direction . An electric field enhancement layer is disposed at least on the inner surface of the groove having an inclined surface inclined at a gradient at least in part and formed in any one of a V-shaped cross-section, a cross-sectional trapezoidal shape, and a cross-sectional polygonal shape. A part or all of the outermost surface formed and in contact with the analyte liquid in the detection groove is Rutotomoni is a detection surface of the target substance, is formed by a plurality parallel to one of said detecting chip has a spacing between grooves of said detection groove adjacent, first a portion forming the spacing between the groove target substance detection plate characterized by Rukoto 1 of the light shielding portion is formed.
<2> The target substance detection plate according to <1>, wherein the plate body is formed of a disk-shaped member.
<3> The plate main body is made of a disk-shaped member, and with respect to the center of the plate, a specimen liquid storage section that stores a specimen liquid disposed at a position closer to the storage section and a cleaning liquid storage section that stores a cleaning liquid A waste liquid storage section that stores waste liquid consisting of the analyte liquid and the cleaning liquid disposed at a position farther from the storage section, and the analyte liquid storage section, the cleaning liquid storage section, and the waste liquid storage section The target substance detection plate according to any one of <1> to <2>, wherein each of the target substance detection plates is connected to the container through a flow path for feeding the analyte liquid, the cleaning liquid, and the waste liquid.
<4> The target substance detection plate according to any one of <1> to <3>, wherein a surface opposite to the surface on which the detection groove of the transparent base portion is formed is formed flat.
<5> The target substance detection plate according to any one of <1> to <4>, wherein the detection groove has a trapezoidal shape in a cross-sectional view.
<6> The target substance detection plate according to <5>, wherein a second light-shielding portion is formed on the bottom surface of the detection groove .
<7> The target substance detection plate according to any one of <1> to <6>, wherein the electric field enhancement layer is formed by arranging a surface plasmon excitation layer that expresses surface plasmon resonance on the groove.
< 8 > The target substance detection plate according to < 7 >, wherein the material for forming the surface plasmon excitation layer includes at least one of gold, silver, copper, platinum, and aluminum.
< 9 > The target substance detection plate according to any one of < 7 > to < 8 >, wherein the surface of the surface plasmon excitation layer is coated with a transparent dielectric.
The <10> electric field enhancing layer, the items <1> which the optical waveguide layer which is formed by a thin film layer and the transparent material formed of a metal material or a semiconductor material over the grooves are formed are arranged in this order <6 > The target substance detection plate according to any one of the above.
< 11 > The target substance detection plate according to < 10 >, wherein the metal material contains at least one of gold, silver, copper, platinum, and aluminum.
< 12 > The target substance detection plate according to < 10 >, wherein the semiconductor material is silicon.
< 13 > The target substance detection plate according to any one of < 10 > to < 12 >, wherein the optical waveguide layer is formed of silica glass.
< 14 > The target substance detection plate according to any one of <1> to < 13 >, wherein a surface treatment for capturing the target substance is performed on the detection surface.
< 15 > Light irradiation for irradiating the electric field enhancement layer with light from the surface opposite to the surface on which the detection groove of the detection chip is formed, and the target substance detection plate according to any one of <1> to < 14 > And a light detection means for detecting fluorescence emitted from a target substance in the sample liquid existing in the detection groove or a fluorescent substance for labeling the target substance based on the irradiation of the light. A target substance detection device.
< 16 > The target substance detection device according to < 15 >, wherein the light irradiation unit includes a light source and a polarizing plate that polarizes light emitted from the light source into linearly polarized light.
< 17 > A target substance detection method for detecting a target substance using the target substance detection apparatus according to any one of < 15 > to < 16 >, wherein the analyte liquid is placed in a flow path of the target substance detection plate. Analyte liquid introduction step of feeding and introducing the analyte liquid into the detection groove of the detection chip, and irradiating the electric field enhancement layer with light from the side opposite to the surface where the detection groove of the detection chip is formed And a light detection step for detecting fluorescence emitted from a target substance in the sample liquid present in the detection groove or a fluorescent substance for labeling the target substance based on the light irradiation. A method of detecting a target substance, comprising:

  According to the present invention, the above-mentioned problems in the prior art can be solved, the number of parts of the detection chip composed of the optical prism and the detection plate used in the SPR sensor and the optical waveguide mode sensor can be reduced, and the size and cost can be reduced. Target substance detection plate, target substance that can detect target substance efficiently, can detect target substance quickly and with high sensitivity, and has a detection chip that allows easy introduction of analyte liquid A detection apparatus and a target substance detection method can be provided.

It is explanatory drawing which shows an example of the optical arrangement | positioning of the SPR sensor using the surface plasmon resonance which concerns on a prior art. It is explanatory drawing which shows an example of optical arrangement | positioning of the optical waveguide mode sensor which concerns on a prior art. It is explanatory drawing explaining the target substance detection plate which concerns on one Embodiment of this invention. It is explanatory drawing partially explaining the target substance detection plate which concerns on one Embodiment of this invention. It is a figure corresponded in the AA sectional view taken on the line of FIG. It is a figure corresponded in the BB sectional drawing of FIG. It is a top view of the target substance detection plate which concerns on other embodiment of this invention. It is explanatory drawing which partially demonstrates the target substance detection plate which concerns on other embodiment of this invention. It is a perspective view which shows the detection chip in this invention. FIG. 8 is a side view of the detection chip shown in FIG. It is explanatory drawing of the detection chip shown in FIG.7 (b). It is sectional drawing which shows the other example of a detection chip. It is sectional drawing which shows another example of a detection chip. It is sectional drawing which shows another example of a detection chip. It is sectional drawing which shows another example of a detection chip. It is explanatory drawing which partially demonstrates the optical arrangement | positioning of the target substance detection apparatus which concerns on one Embodiment of this invention. It is explanatory drawing which shows the optical system produced for the effectiveness check of a detection chip. It is a graph which shows the wavelength dependence of the transmitted light intensity when irradiating p-polarized linearly polarized light and s-polarized linearly polarized light.

(Target substance detection device and target substance detection plate)
The target substance detection apparatus of the present invention includes the target substance detection plate of the present invention, a light irradiation means, a light detection means, and other members as required.
In the target substance detection apparatus of the present invention, for example, biological substances such as viruses, proteins, DNA, biomarkers, contaminants, poisons, deleterious substances, and other various molecules can be detected as target substances.

<Target substance detection plate>
The target substance detection plate has a translucent plate main body and a detection chip for detecting the target substance.

-Plate body-
The plate main body is formed with one or a plurality of concave accommodating portions for accommodating the detection chip, and a flow path for supplying an analyte liquid for verifying the presence of the target substance to the accommodating portion.

There is no restriction | limiting in particular as a shape of the said plate main body, According to the objective, it can select suitably, For example, plate members, such as disk shape, triangular plate shape, and square plate shape, are mentioned.
The material for forming the plate body is not particularly limited as long as it has translucency, and can be appropriately selected according to the purpose. For example, plastic materials such as cyclic polyolefin, acrylic, polystyrene, and polycarbonate, and high transparency are available. The glass material which can be ensured is mentioned.

A method for forming the housing portion is not particularly limited and may be appropriately selected depending on the purpose. For example, a method of forming the plate body by injection molding, or mechanical processing such as cutting is performed on the plate body. And the like.
There is no restriction | limiting in particular as a shape of the recessed part of the said accommodating part, According to the shape and magnitude | size of the said detection chip to accommodate, it can select suitably.
The bottom surface of the recess is preferably formed as a flat surface so as to be in stable contact with one surface of the detection chip to be accommodated.

The method for forming the flow path is not particularly limited and may be appropriately selected depending on the purpose. For example, the plate body is formed by injection molding, or the plate body is subjected to mechanical processing such as cutting. And the like.
There is no restriction | limiting in particular as a planar shape when the said plate main body of the said flow path is seen from a plane, According to the objective, it can select suitably, It can be linear form, curved form, etc. For example, when the plate body has a disc shape, the plate body may have a curved shape along the rotation direction.
Moreover, as the cross-sectional shape, for example, a concave shape, a V-shape, a semicircular shape, a semi-elliptical shape, and a trapezoidal shape can be used in the cross-section.

  The plate body is not particularly limited, and further includes a sample liquid storage unit for storing the sample liquid, a cleaning liquid storage unit for storing a cleaning liquid for cleaning the sample liquid, the sample liquid, and the cleaning liquid. You may have the waste liquid storage part which stores the waste liquid which becomes. Moreover, you may have a cover part for preventing that these liquids spill. It is also preferable to form a vent hole for venting the air in the reservoir so that the liquid smoothly enters these reservoirs.

-Detection chip-
The detection chip is housed in the housing portion and has a transparent base portion and a detection groove.
The detection chip is accommodated so that the surface of the transparent base portion opposite to the surface on which the detection groove is disposed is bonded to the bottom surface of the accommodation portion.
The detection chip may be housed in a fixed state with respect to the housing part or may be housed without being fixed.
When not fixed, it is preferable to restrict the position of the detection chip so that the position of the detection chip does not fluctuate in the housing portion. For example, the housing part is cut into a rectangular column shape or an elliptical column shape, and the position is regulated by arranging the detection chip having a rectangular column shape or an elliptical column shape slightly smaller than the size of the housing part therein.

--Transparent substrate part--
The transparent base portion is a plate-like member that transmits light.
The material for forming the transparent base portion is not particularly limited as long as it has the light transmission property and the detection groove can be formed, and can be appropriately selected according to the purpose. A plastic material such as polystyrene or polycarbonate that can be mass-produced by use, or a glass material such as silica glass that can ensure high transparency is preferable.

The transparent base portion functions as an optical prism used in a conventional SPR sensor or an optical waveguide mode sensor.
That is, it plays a role of introducing light emitted from the light irradiation means at a specific incident angle at which surface plasmon resonance or an optical waveguide mode is excited with respect to an inclined surface of a groove portion described later formed in the detection groove. Have.
Therefore, the lower limit of the refractive index of the transparent base portion is preferably 1.33 or more, more preferably 1.38 or more, and further preferably 1.42 or more. Further, the upper limit of the refractive index is preferably 4 or less, and more preferably 3 or less.
The refractive index will be described later using a separate drawing.

  The transparent base portion is disposed in the housing portion such that a surface opposite to the surface on which the detection groove is formed is in contact with or close to the bottom portion of the housing portion. This surface is preferably formed flat because it is an incident surface for light irradiated from the bottom direction of the housing portion.

--Detection groove--
The detection groove is formed on one surface of the transparent base portion, and is formed as a groove that is connected to the flow path and into which the analyte liquid is introduced. In addition, the detection groove has an electric field enhancement layer on an inner surface of the groove part formed so as to have at least a part of an inclined surface inclined with a single inclination with respect to one surface of the transparent base part in a cross-sectional view. Is formed. Here, the electric field enhancement layer includes a layer formed with a layered structure capable of exciting surface plasmon resonance (surface plasmon excited layer) and a layer formed with a layered structure capable of exciting optical waveguide mode. Point to. The electric field enhancement layer will be described later separately.

The number of the detection grooves is not particularly limited and may be appropriately selected according to the purpose. The number of detection grooves may be one or plural, and the detection groove may be a detection for detecting the target substance. Therefore, it is desirable to increase the area as much as possible to improve the detection sensitivity of the target substance.
In this case, it is preferable that a plurality of the detection grooves are formed in parallel for one detection chip.

--Groove-
The method for forming the groove is not particularly limited and may be appropriately selected depending on the purpose. For example, a method of injection molding the transparent base portion so as to have the groove, Examples thereof include a method of forming using a means such as a cutting means.

The groove has at least a part of an inclined surface that is inclined with a single gradient with respect to one surface of the transparent base portion in a cross-sectional view.
The shape of the groove is not particularly limited as long as it has at least a part of the inclined surface, and examples thereof include a V-shaped cross section, a trapezoidal cross section, and a polygonal cross section. However, the inclined surface is a curved surface, and does not include a U-shaped cross section or a semicircular cross-sectional shape that does not have any portion inclined with a single gradient. When the inclined surface is a curved surface, the excitation of the surface plasmon or the optical waveguide mode in the electric field enhancement layer is limited, and the target substance cannot be sufficiently detected.
Accordingly, at least a part of the inclined surface inclined with the one gradient needs to be present on the side surface of the groove constituting the groove portion. On the other hand, from this point of view, the inclined surface only needs to be formed on the detection surface for detecting the target substance, and need not be formed over the entire length of the groove.
Further, even when the inclined surface is formed over the entire length of the groove portion, it is not necessary to use all of the inclined surface for detection, and only a part thereof is irradiated with light, or Only a part of the target substance may be captured and detected.
As long as it has such an inclined surface as a groove side surface which comprises the said groove part, there is no restriction | limiting, You may form symmetrically and may be formed asymmetrically.
Details of these will be described later with reference to the drawings.

The opening width when the groove is viewed from one surface of the transparent base portion, that is, the distance between the side surfaces of the left and right groove portions on the one surface is not particularly limited, but is preferably 5 μm to 5 cm. If the opening width is less than 5 μm, the structure is fine, making it difficult to manufacture and increasing the manufacturing cost. If the groove is small, the detection groove becomes narrow and a highly viscous liquid does not flow. End up. On the other hand, when the opening width exceeds 5 cm, the inner volume of the detection groove increases accordingly, and thus a lot of analyte liquid is required.
Moreover, there is no restriction | limiting in particular as the depth of the said groove part, However For the same reason as the above, 5 micrometers-5 cm are preferable.
Further, when a plurality of the detection grooves are arranged, that is, when a plurality of the groove portions are formed, the interval between the adjacent groove portions is not particularly limited, but is preferably 5 μm to 5 cm. If the distance is less than 5 μm, the structure is fine, making it difficult to manufacture and increasing the manufacturing cost. If the distance exceeds 5 cm, the detection chip itself becomes large, which may cause inconveniences such as requiring a large amount of material during production and taking up space during storage.
In addition, light emitted from the light irradiating means is directly emitted to the light detecting means side at the portions forming the interval between the groove portions. Therefore, light shielding portions for attenuating the light are provided at these portions. Is preferred.

---- Electric field enhancement layer ---
There is no restriction | limiting in particular as said electric field enhancement layer, According to the objective, it can select suitably, For example, (A) Forming by arranging the surface plasmon excitation layer which expresses surface plasmon resonance on the said groove part In addition, (B) a layer structure for exciting the optical waveguide mode may be disposed on the groove. Here, the layer structure for exciting the optical waveguide mode is formed by arranging a thin film layer formed of a metal material or a semiconductor material and an optical waveguide layer formed of a transparent material in this order on the groove. be able to.

(A) The material for forming the surface plasmon excitation layer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include metal materials having a negative dielectric constant at the wavelength of incident light. A metal material containing at least one of gold, silver, platinum and aluminum is preferable.
When the metal layer formed of the metal material receives light at a certain incident angle through the prism, the evanescent wave that oozes to the surface side satisfies the excitation condition of the surface plasmon, and the surface plasmon is formed on the surface of the metal layer. Resonance is developed.
The optimum thickness of the metal layer is determined by the metal material and the wavelength of incident light. It is known that this value can be calculated from calculation using the Fresnel equation. Generally, when surface plasmon is excited from the near ultraviolet region to the near infrared region, the thickness of the metal layer is several nm to several tens of nm.

The method for forming the surface plasmon excitation layer, that is, the metal layer is not particularly limited, and examples thereof include well-known formation methods such as a vapor deposition method, a sputtering method, a CVD method, a PVD method, and a spin coating method. In the case where the material for forming the transparent base portion on which is formed is the plastic material or the glass material, if the metal layer is directly formed on the groove portion, the adhesion may be low and 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 inner surface of the groove and form the metal layer on the adhesive layer.

When detecting light emission from a target substance or a fluorescent substance that labels the target substance described later, when the target substance or the fluorescent substance comes close to the metal layer, the emitted light is absorbed again by the metal layer, A phenomenon called quenching occurs in which the luminous efficiency decreases.
In this case, if a coating layer having a thickness of several nanometers to several tens of nanometers is formed for the purpose of separating the target substance and the fluorescent substance from the surface of the metal layer, quenching is suppressed and a decrease in luminous efficiency can be suppressed. It has been known.
Therefore, it is preferable that the surface plasmon excitation layer, that is, the surface of the metal layer is covered with a transparent dielectric.
The transparent dielectric is not particularly limited, and may be a material capable of forming a transparent film having a thickness of several nanometers to several tens of nanometers, such as a glass material such as silica glass, an organic polymer material, or a protein such as bovine serum albumin. Can be mentioned.

(B) In the formation of the layer structure for exciting the optical waveguide mode, the metal material for forming the thin film layer is not particularly limited, and examples thereof include generally available and stable metals and alloys thereof. However, it preferably contains at least one of gold, silver, copper, platinum and aluminum.
There is no restriction | limiting in particular as a semiconductor material which forms the said thin film layer, For example, semiconductor materials, such as silicon and germanium, or a known compound semiconductor material are mentioned, However, Since silicon is cheap and easy to process, it is preferable.
The thickness of the thin film layer is the same as that of the metal layer of the surface plasmon excitation layer, and the optimum value is determined by the material of the thin film layer and the wavelength of incident light, and this value is calculated using the Fresnel equation. It is known that calculation is possible from calculation. Generally, when using light in a wavelength band from the near ultraviolet region to the near infrared region, the thickness of the thin film layer is several nm to several hundred nm.
When the metal material is selected as the thin film layer, it is preferable to dispose the aforementioned chromium or nickel adhesive layer between the groove and the thin film layer in order to improve adhesion.

  In addition, the material for forming the optical waveguide layer is not particularly limited as long as it is a transparent material having high light transmittance. For example, a resin material such as silicon oxide, silicon nitride, and acrylic resin, a metal oxide such as titanium oxide, Examples thereof include metal nitrides such as aluminum nitride, but silicon oxide is preferable because it is easy to manufacture and has high chemical stability. In this case, if the thin film layer is formed of silicon, it can be easily formed by oxidizing the surface side of the silicon layer.

---- Surface treatment ---
When the target substance is selectively detected, there is no particular limitation, but it is preferable that a surface treatment for specifically capturing the target substance is performed on the surface of the detection groove, that is, the detection surface.
The surface treatment method is not particularly limited and can be appropriately selected depending on the purpose.For example, when the noble metal is used for the metal layer as the surface plasmon excitation layer, Examples include a chemical modification method in which a capture substance is immobilized on the detection surface using a metal-thiol bond, and a glass material such as silica glass is used as the transparent dielectric covering the metal layer. When the layer is used as the detection surface, a chemical modification method for immobilizing a capture substance on the detection surface using silane coupling may be used.
The surface treatment method when the surface of the optical waveguide layer is the detection surface is not particularly limited and can be appropriately selected according to the purpose.For example, using silicon oxide as the optical waveguide layer, When the surface is used as the detection surface, a chemical modification method for immobilizing a capture substance on the detection surface by silane coupling as in the case of the glass coating layer.

Here, an embodiment of the target substance detection plate will be described with reference to FIGS.
As shown in FIG. 3, the plate body 2 of the target substance detection plate 1 is formed in a disc shape and can be rotated in the direction of arrow A in the figure by a rotating means such as a spindle (not shown).
As shown in the enlarged portion, the plate body 2 is formed with a flow path 3, an accommodating portion 4, and an analyte liquid storage portion 5 for accumulating the analyte liquid. A sample liquid is introduced from the liquid storage section 5 into the storage section 4 via the flow path 3. 4 'and 5' are waste liquid storage units for storing the waste liquid.
In addition, a detection chip 8 is stored in the storage unit 4 to detect a target substance present in the sample liquid. That is, as shown in FIG. 4, the accommodating part 4 is formed in a concave shape, and the detection chip 8 is accommodated therein. The container 4 is connected to the flow path 3 on the side surface thereof, and the analyte liquid can be sent to the detection chip 8. Note that reference numeral 9 in the figure denotes a lid portion disposed for the purpose of preventing the sample liquid from spilling from the storage portion 4.

A state in which the detection chip 8 is accommodated in the accommodating portion 4 will be described with reference to FIGS. FIG. 5A is a view corresponding to a cross section taken along line AA of FIG. FIG. 5B is a view corresponding to a cross section taken along line BB in FIG.
As shown in these drawings, the detection chip 8 is accommodated in the accommodating portion 4. The analyte liquid sent from the flow path 3 is introduced into the detection groove 6. Light L is irradiated from the light source 10 installed outside the plate body 2 from the surface of the transparent base portion 7 opposite to the surface on which the detection groove 6 is formed, and the light enters the detection groove 6. When the detection groove 6 is irradiated with light, the electric field is enhanced by the electric field enhancement layer disposed in the detection groove 6, and the fluorescence from the target substance or the fluorescent substance that labels the target substance can be observed. The target substance may be detected in a state where the sample liquid is introduced into the detection groove 6, but after the sample liquid is introduced, the target substance contained in the sample liquid is immobilized on the detection surface. It is preferable to carry out the cleaning after introducing the cleaning liquid into the container 4 after washing with the substance and washing the impurities and impurities because signals due to the impurities and impurities can be removed.
Here, after the analyte liquid supplied from the flow path 3 on the side of supplying the analyte liquid to the container 4 in the plate body 2 flows along the detection groove 6 of the detection chip 8, the waste liquid reservoir 5 ′. It is preferable that the detection chip 8 is disposed in the accommodating portion 4 so as to be discharged to the flow path 3 connected to the. In order to realize this, the sample liquid supply port to the storage unit 4, that is, the junction between the flow channel 3 on the side for supplying the sample liquid to the storage unit 4 and the storage unit 4, An outlet for discharging the sample liquid, that is, a straight line connecting the flow path 3 for discharging the waste liquid from the storage portion 4 and the joint portion of the storage portion 4, and the detection groove 6 are arranged in parallel or from the parallel state. The deviation is preferably within ± 45 ° in terms of angle. When the flow path 3 and the detection groove 6 are connected in this way, the analyte liquid can be efficiently introduced from the flow path 3 to the detection groove 6. In addition, the cleaning liquid can be easily introduced into the detection groove 6, and the analyte liquid remaining in the detection groove 6 can be easily cleaned. In the example shown in FIGS. 5A and 5B, the detection groove 6 and the straight line connecting the sample liquid supply port to the storage unit 4 and the discharge port for discharging the sample liquid from the storage unit 4 are parallel. Is arranged.

  In the target substance detection plate 1 configured as described above, a plurality of detection structures each including the flow path 3 and the accommodating part 4 are formed. Can be detected. Moreover, different target substances can be detected for each detection structure, and a plurality of target substances can be detected in one operation, and an efficient detection test can be performed. Further, since the detection groove 6 of the detection chip 8 is configured to play the roles of the flow path of the analyte liquid and the detection surface of the target substance in the accommodating portion 4, the flow path and the detection surface are separately provided. There is no need to manufacture, and the production cost can be reduced.

Another embodiment of the target substance detection plate will be described with reference to FIGS. 6 (a) and 6 (b).
As shown in FIG. 6A, the target substance detection plate 100 is composed of a disc-shaped plate body 102. The plate body 102 includes a housing portion 104 that houses a detection chip 108, and a subject that stores a specimen liquid. A sample liquid storage unit 105, a cleaning liquid storage unit 106 for storing a cleaning liquid for cleaning the sample liquid, a waste liquid storage unit 107 for storing a waste liquid composed of the sample liquid and the cleaning liquid, a storage unit 104, and a sample liquid storage unit 105 , A flow path 103b that connects the storage section 104 and the cleaning liquid storage section 106, and a flow path 103c that connects the storage section 104 and the waste liquid storage section 107. The central part of the plate main body 102 is cut out to have a shape like a commercially available CD.
The analyte liquid storage unit 105 and the cleaning liquid storage unit 106 are disposed at a position closer to the center of the plate body 102 than the storage unit 104, and the waste liquid storage unit 107 is disposed at a position farther from the storage unit 104.
FIG. 6B shows an enlarged view of one detection unit including the storage unit 104, the analyte liquid storage unit 105, the cleaning liquid storage unit 106, the waste liquid storage unit 107, and the flow paths 103a to 103c.
The detection chip 108 accommodated in the accommodation unit 104 has one detection groove 109. The flow paths 103a and 103b are formed in a substantially Y shape with respect to the detection groove 109. At this time, the detection groove 109 and the straight line connecting the joint portion between the storage portion 104 and the flow paths 103a and 103b and the joint portion between the storage portion 104 and the flow path 103c are arranged with a deviation of ± 22.5 ° from parallel. ing. Other configurations are appropriately configured according to the configuration of the target substance detection plate 1.

According to such a target substance detection plate 100, when the plate body 102 is rotated, the analyte liquid stored in the analyte liquid storage unit 105 can be sent to the storage unit 104 by centrifugal force. The cleaning liquid stored in the cleaning liquid storage unit 106 can be sent to the storage unit 104, and the analyte liquid and the cleaning liquid sent to the storage unit 104 can be sent to the waste liquid storage unit 107. Further, the analyte liquid and the cleaning liquid can be easily introduced into the detection groove 109 of the detection chip 108, and the detection test and the cleaning can be performed efficiently.
Although an example in which one detection groove 109 is formed on the detection chip 108 is shown in the drawing, a plurality of detection grooves 109 may be formed on one detection chip 108. Further, as shown in FIG. 6A, one detection unit including a storage unit 104, a specimen liquid storage unit 105, a cleaning liquid storage unit 106, a waste liquid storage unit 107, a detection chip 108, and flow paths 103a to 103c. It is preferable to form a plurality of units on the plate body 102 because a plurality of detection tests can be performed simultaneously.

Next, an example of an embodiment of the detection chip will be described with reference to the drawings.
The detection chip 11 according to an embodiment of the present invention shown in FIG. 7A is configured by forming a detection groove 13 having a V-shaped cross section on one surface of the transparent base portion 12. Further, as shown in FIG. 7B for explaining the cross section of the detection groove 13, the electric field enhancing layer 14 is formed on the inner surface of the groove portion formed in a V-shaped cross section. An analyte liquid for verifying the presence of the target substance is introduced into the detection groove 13, and its outermost surface, here, the surface of the electric field enhancement layer 14 is used as the target substance detection surface.
In the electric field enhancement layer 14, surface plasmon resonance or an optical waveguide mode is excited by light irradiated from a light irradiation means (not shown), and a strong electric field is formed in the layer and in the vicinity of the layer surface. In the said light irradiation means, light is irradiated from the surface R side which is a surface opposite to the surface in which the detection groove | channel 13 of the transparent base | substrate part 12 is formed.

  At this time, as shown in FIG. 7C, which is an explanatory diagram of FIG. 7B, the transparent base portion 12 has a region functioning as a triangular prism indicated by a dotted line, and the light L irradiated from the surface R side. Is incident on the electric field enhancement layer 14 at a specific incident angle. That is, the transparent substrate 12 functions as an optical prism in the SPR sensor and the optical waveguide mode sensor, and enhances the electric field in the vicinity of the surface of the electric field enhancement layer 14 to enable detection of a target substance derived from this phenomenon. . In addition, since the surface R functions as an incident surface of the prism, it is preferable that the surface R has high flatness.

The base angle φ at the groove portion of the detection groove 13 shown in FIG. 7C is determined by the incident angle θ of the light L with respect to the inclined surface forming the groove portion. For example, in the example of the detection chip 11 shown in FIG. 7C, the surface on which the detection groove 13 of the transparent base portion 12 is formed and the surface R which is the opposite surface are parallel to each other, and two grooves forming the groove portion are formed. The inclined surfaces are bilaterally symmetric and light L is incident perpendicularly to the surface R from the light irradiation means. In this case, the base angle φ (°) is selected as an angle of (90 ° −θ) × 2.
Since the incident angle θ is determined by the excitation conditions of the surface plasmon resonance and the optical waveguide mode, the base angle φ depends on the refractive index of the material forming the transparent base portion 12 and the configuration of the electric field enhancement layer 14.
Here, if the refractive index of the transparent base portion 12 is too low, it is necessary to increase the incident angle θ, and consequently the base angle φ. In the case of a micro-channel having an opening width of the detection groove 13 of several hundreds μm or less, since the formation of the detection groove 13 becomes difficult when the base angle φ is 30 ° or less, the refractive index of the transparent base portion 12 is 1.38 or more is preferable, and 1.42 or more is more preferable. On the other hand, in the case of a detection groove having such a size that the processing difficulty is not particularly problematic, the refractive index of the transparent base portion 12 may be lower, but is preferably at least higher than the refractive index of water 1.33.
On the other hand, if the refractive index of the transparent base portion 12 is too high, there is a problem that when the light L is incident on the surface R, reflection on the surface R becomes strong. In addition, candidates for materials having a high refractive index and high transparency are limited. Therefore, the refractive index of the transparent base portion 12 is preferably 4 or less, and more preferably 3 or less.

In this example, as shown in FIGS. 7A and 7B, the detection groove 13 is formed in parallel in the detection chip 11. By forming in this way, the surface area of the detection surface can be increased as compared with the case where there is one detection groove, and the detection sensitivity of the target substance can be improved.
Further, as described above, there may be a space 15 between the groove portions of the adjacent detection grooves. When the transparent base portion 12 is injection-molded, the groove portion formed so as to have such a gap 15 needs to be formed at an acute angle in the stamper groove portion that forms this groove shape, that is, the portion facing the gap 15. The production cost can be reduced.
Further, as described above, it is preferable to provide a light shielding portion for attenuating light at the interval 15.

  FIG. 8 shows another embodiment of the detection chip. The detection chip 21 has a transparent base portion 22 and a plurality of detection grooves 23. The shape of the detection groove 23 is a trapezoid in a cross-sectional view. According to such a detection chip, the bottom of the groove is formed flat rather than an acute angle as compared with a groove having a V-shaped cross section. Therefore, when cleaning is performed after the detection test is finished, a cleaning liquid is applied to the bottom of the groove. Is easy to flow in and can be efficiently cleaned, which is preferable. However, it is preferable to provide a light-shielding part for attenuating light in such a flat part as in the case of the interval 15 so that light from the light irradiation means does not reach the photodetector side as much as possible.

In the detection groove, the two inclined surfaces constituting the groove may not be symmetrical.
For example, as shown in FIG. 9, the detection groove 33 may be formed so that the two inclined surfaces have different gradients. In this case, since different light emission characteristics are obtained on the respective inclined surfaces, two different detections can be performed simultaneously.
For example, the left and right surfaces are set so that electric field enhancement occurs at different wavelengths. For example, the left surface is set so that surface plasmons are excited by light of 550 nm, and the right surface is set so that surface plasmons are excited by light of 660 nm. On the left side, the sample is measured so that the fluorescent dye emitting with 550 nm excitation light is specifically adsorbed. On the right side, the fluorescent dye emitting with 660 nm excitation light is specifically adsorbed. Measure the sample. The light source is provided with excitation light of 550 nm and excitation light of 660 nm, respectively, and signals by each excitation light can be detected from the left and right surfaces by alternately irradiating or shielding with a filter or the like. Two specimens can be detected simultaneously. In FIG. 9, reference numeral 31 indicates a detection chip, and reference numeral 32 indicates a transparent substrate portion.

The two inclined surfaces may be formed with a multi-stage gradient.
For example, as shown in FIG. 10, the detection groove 43 may be formed so that the two inclined surfaces have a multi-stage gradient. In this case, it is possible to simultaneously perform detection with different excitation wavelengths for each gradient. In addition, in FIG. 10, the code | symbol 41 shows a detection chip and the code | symbol 42 shows a transparent base | substrate part.

  In addition, in the case where fluorescence is detected only on one inclined surface, the angle formed by the surface not used for detection does not particularly affect the detection. As shown in FIG. 11, the surface not used for detection of the detection groove 53 may be formed vertically. In FIG. 11, reference numeral 51 denotes a detection chip, and reference numeral 52 denotes a transparent substrate portion.

<Light irradiation means>
The light irradiation means is means for irradiating the electric field enhancement layer with light from a surface side of the detection chip opposite to a surface where the detection groove is formed.
There is no restriction | limiting in particular as a structure of the said light irradiation means, According to the objective, it can select suitably, For example, light sources, such as a laser, a white lamp, LED, the collimator which collimates the light from the said light source, From the said light source It can be configured by appropriately selecting from known optical members such as a lens for condensing the light of the above and a polarizing plate for polarizing the light from the light source.
Especially, it is preferable to have a polarizing plate that polarizes light emitted from the light source into linearly polarized light.

<Light detection means>
The light detection means detects fluorescence emitted from the target substance or the fluorescent substance that labels the target substance in the sample liquid present in the detection groove by light emitted from the light irradiation means. It is configured as a means to do.

The configuration of the light detection means is not particularly limited and may be appropriately selected according to the purpose. The photodetector for detecting the fluorescence, such as a CCD, a photodiode, or a photomultiplier tube, and the fluorescence to the light An optical fiber that leads to a detector and a condensing lens that condenses the fluorescence and guides it to the photodetector can be appropriately selected from known optical members.
In addition, in order to distinguish whether the detected light is derived from the fluorescence emitted from the target substance or the fluorescent substance or from other light, only the light in the fluorescence wavelength band is transmitted. Detection by the photodetector may be performed via a wavelength filter.

<Other members>
There is no restriction | limiting in particular as said other member, According to the objective, it can select suitably, For example, a liquid feeding pump etc. are mentioned. Examples of the liquid delivery pump include a pump that delivers the analyte liquid to the flow path.

  FIG. 12 shows a configuration example of the target substance detection apparatus that detects fluorescence emitted from the target substance or the fluorescent substance. Here, the detection chip 61, the light irradiation means (not shown) that irradiates the light L from the surface R side of the detection chip 61, and the fluorescence k emitted from the target substance or the fluorescent substance is the light in the wavelength band of the fluorescence k. And a photodetector 67 that detects light through a wavelength filter 68 that transmits only the light. Further, the detection chip 61 has a transparent base portion 62 and a detection groove 63 formed on one surface thereof.

As the light L to be irradiated, laser light corresponding to the wavelength of the excitation band of the target substance or the fluorescent dye, or light monochromatized by an optical filter or a spectroscope is used.
At this time, the light irradiation means is rotated so as to draw a semicircle on the surface R side of the detection chip 61, or the detection chip 61 is rotated around the fixed light irradiation means, and the light L When the incident angle is changed and the electric field enhancement layer of the detection groove 63 is irradiated with the light L, the phenomenon that the emission intensity increases can be observed and observed at a specific angle at which the surface plasmon resonance or the optical waveguide mode is excited. Whether the light emission is caused by surface plasmon resonance or optical waveguide mode, or the stray light of light L that is not involved in excitation of surface plasmon resonance or optical waveguide mode is irradiated to the fluorescent material, and emitted regardless of the detection of the target substance It can be confirmed.

  However, since the incident angle changing mechanism and the rotating mechanism for rotating the detection chip 61 require a movable part, there is a demerit that the detection apparatus itself is increased in size. In order to configure the apparatus in a small size and at a low cost, a method of observing the fluorescence intensity and detecting the target substance with the incident angle fixed at a constant angle is preferable.

When the target substance itself emits fluorescence k, the target substance is captured on the detection surface of the detection groove 63, and the presence / absence and amount of the target substance are observed by observing the presence / absence and emission intensity of the target substance. it can.
However, since many substances do not exhibit strong light emission characteristics, after capturing on the detection surface of the detection groove 63, the fluorescent substance is attached to the target substance and light emission is observed.
The method for attaching the fluorescent substance is not particularly limited, and a known technique can be applied. For example, the fluorescent substance is bound to an antibody that specifically adsorbs to the target substance, and the fluorescent substance is used. And a method of adsorbing the attached antibody to the target substance.

(Target substance detection method)
The target substance detection method of the present invention is a method for detecting the target substance using the target substance detection apparatus of the present invention, and includes an analyte liquid introduction step, a light irradiation step, and a light detection step.

The analyte introducing step is a step of feeding the analyte liquid into the flow path of the target substance detection plate and introducing the analyte liquid into the detection groove of the detection chip.
The light irradiation step is a step of irradiating the electric field enhancement layer with light from the surface opposite to the surface on which the detection groove of the detection chip is formed.
The light detection step is emitted from the target substance in the sample liquid existing in the detection groove or a fluorescent substance that labels the target substance based on the light irradiation performed in the light irradiation step. This is a step of detecting fluorescence.
These steps can be appropriately performed according to the matters described in the target substance detection apparatus.

Example 1
In order to confirm the effectiveness of the present invention, detection chip 71, light irradiation means (not shown) for irradiating light L from the surface R side of detection chip 71, and fluorescence emitted from the target substance or the fluorescent substance are detected. A prototype having an optical detector 77 was manufactured (see FIG. 13).

Here, the detection chip 71 was manufactured as follows.
First, a plate-like transparent base portion 72 having a V-shaped cross section formed by injection molding using polystyrene as a forming material was produced. The two inclined surfaces constituting the groove were left-right symmetric, and the base angle φ was 49 °. Moreover, the opening width of the groove was 300 μm.
Next, with respect to the surface of the transparent base portion 72 on which the groove is formed, chromium is applied so that the thickness in the flat region is 0.6 nm from a direction perpendicular to the flat region where the groove is not formed. A chromium thin film 74a as an adhesive layer was formed on the entire surface where the groove was formed by vapor deposition.
Next, gold was vapor-deposited so as to have a thickness of 100 nm in the flat region, and a gold thin film 74b as a surface plasmon excitation layer was formed on the chromium thin film 74a.
Next, a silica glass thin film was deposited by sputtering so that the thickness in the flat region was 49 nm, and the surface of the gold thin film 74b was covered with a transparent dielectric 74c.
As a result, a detection groove 73 having a groove shape substantially the same as the shape of the groove portion was formed in the transparent base portion 72. At this time, the chrome thin film 74a and the gold thin film 74b laminated on the upper surface of the transparent base portion 72 other than the opening of the detection groove 73 serve as a light shielding portion.
Thus, the detection chip 71 was manufactured.

As shown in FIG. 13, the detection chip 71 is filled with water and irradiated with light from the light irradiating means, and from the surface R side of the detection chip 71 in a direction perpendicular to the surface R. Light was incident with a beam diameter of about 1 mm in diameter. The light irradiation means includes a white light source and a polarizing plate that linearly polarizes light emitted from the white light source into p-polarized light, and linearly s-polarizes light emitted from the white light source and the white light source. It comprised with the polarizing plate made to polarize, and comprised it in two ways.
The transmitted light of the white light irradiated from the R surface side of the detection chip 71 by the two types of light irradiation means is measured by the photodetector 77 arranged opposite to the surface where the detection groove 73 of the detection chip 71 is formed. did. FIG. 14 shows the result.
FIG. 14 shows the wavelength dependence of transmitted light intensity, and a clear increase in transmitted light intensity can be confirmed in the wavelength region of 570 nm to 870 nm in the case of p-polarized light as compared with the case of s-polarized light. Considering that surface plasmons cannot be excited in the case of s-polarized light, this phenomenon is caused by excitation of surface plasmons in the wavelength region by p-polarized incident light, resulting in strong scattering of the incident light. This is thought to have occurred.
Thus, if the detection chip 71 is used, it is possible to easily excite surface plasmons with the detection chip 71 without going through a complicated bonding process between the prism and the detection chip as in the prior art. The fluorescence from the fluorescent substance can be easily enhanced by the excitation of the surface plasmon. Further, if the target substance detection plate that accommodates the detection chip 71 is used, the target substance can be efficiently detected.

(Example 2)
In the same manner as in Example 1, first, a plate-shaped transparent base portion 72 having a V-shaped cross section formed by injection molding using polystyrene as a forming material was produced. The structure of the groove is the same as in the first embodiment. Chromium is vapor-deposited on the surface of the transparent substrate 72 where the groove is formed, from the direction perpendicular to the flat area where the groove is not formed, so that the thickness of the flat area is 0.6 nm. Then, a chromium thin film layer 74a as an adhesive layer was formed. Next, gold was deposited on the chromium layer so that the thickness in the flat region was 120 nm, thereby forming a gold thin film layer 74b as a surface plasmon excitation layer. Next, a silica glass thin film (transparent dielectric layer 74c) was deposited on the gold layer by sputtering so that the thickness in the flat region was 49 nm. As a result, the detection groove 73 was formed in the transparent base portion 72.
Thereafter, the transparent substrate on which the thin film was deposited was immersed in a weak alkaline aqueous solution for 24 hours and then dried, and then immersed in an ethanol solution of 0.1 v / v% 3-aminopropyltriethoxysilane for 15 hours to react on the silica glass surface. The amino group was modified. Thereafter, the transparent substrate is rinsed with ethanol, dried, and phosphate buffered saline containing 0.5 mM sulfosuccinimidyl-N- (D-biotinyl) -6-aminohexanate is dropped onto the detection groove 73. Then, it was allowed to stand at room temperature for 2 hours, and biotin was introduced as a substance for capturing the target substance on the detection groove surface. Thus, the detection chip 71 was manufactured.

Next, the target substance detection plate 100 shown in FIG. 6A was produced as follows.
A COP (cyclic polyolefin) substrate is used as a base material for forming the plate body 102, and NC (Numerical Control) is appropriately replaced with a cutting tool having a diameter of 0.01 to 4 mm based on the CAD design. Cutting was performed using a processing machine, and the plate main body 102 including the storage unit 104, the specimen liquid storage unit 105, the cleaning liquid storage unit 106, the waste liquid storage unit 107, and the flow paths 103a to 103c was manufactured.
The shape of the accommodating portion 104 was a cylindrical shape having a diameter of 5.2 mm and a depth of 1.6 mm.
The above-described detection chip 71 (chip plate thickness: 1.5 mm) was cut into a cylindrical shape with a diameter of 5.2 mm and incorporated by machining with an NC processing machine.
At this time, the back surface of the detection chip 71 is assembled in the housing portion 104 after the edge is dropped so that the assembly is easy.
Thereafter, the entire surface of the plate body 102 was sealed (covered) with a pressure-sensitive adhesive transparent sheet so as to cover all the flow paths 103a to 103c. Next, a part of the seal was removed using a CO ₂ laser marker for the purpose of injecting the analyte liquid or for the purpose of air venting.
Thereafter, when the boundary surface of the incorporated detection chip 71 was observed with a confocal microscope, the gap with the surface of the plate body 102 (that is, the back surface of the seal) was 50 μm. The analyte is introduced into the gap and the fluorescent label attached to the target substance adsorbed on the inner wall of the detection groove 73 emits light strongly due to the electric field enhancement effect, so that the target substance can be detected with high sensitivity. is there. The gap is preferably as narrow as about 0 to 200 μm. This is because the antigen-antibody reaction is accelerated by thinning this part, and detection in a short time becomes possible.
The flow path 103a from the analyte liquid storage section 105 to the storage section 104 has a width of 500 μm and a depth of 100 μm, and the flow path 103b from the cleaning liquid storage section 106 to the storage section 104 has a width of 200 μm and a depth of 50 μm. The flow path 103c from the storage unit 104 to the waste liquid storage unit 107 has a width of 30 μm and a depth of 50 μm.
As the solution to be detected, phosphate buffered saline containing 100 nM streptavidin with a fluorescent dye Alexa700 (manufactured by Invitrogen) as a target substance was used. This detection liquid was injected into the detection groove 73 via the flow path 103a, and after filling the detection groove 73 with the detection liquid, it was left at room temperature for 1 hour to capture streptavidin with biotin.
Thereafter, in order to clean impurities, phosphate buffered saline containing 0.05 v / v% Triton X-100 (manufactured by Nacalai Tesque) was injected into the detection groove 73 through 103b and washed. The detection groove 73 was filled with phosphate buffered saline.
The target substance detection plate 100 that has undergone the above process was irradiated with light using an LED with an optical filter that emits light having a wavelength of 680 nm ± 10 nm provided with a collimator lens and a polarizing plate. In addition, a cooled CCD camera is used as the photodetector 77, and an optical filter that transmits light with a wavelength of 710 nm or more and an optical filter that transmits light with a wavelength of 720 nm or more are installed in front of the CCD camera, Configured. The exposure time was 60 seconds.
When p-polarized light was irradiated from the light irradiation means, fluorescence from Alexa 700 could be observed. On the other hand, when s-polarized light was irradiated from the light irradiation means, fluorescence from Alexa 700 was not observed. Since the surface plasmon is excited only by irradiation with p-polarized light, the observation results show that the fluorescence from the fluorescent dye is enhanced by excitation of the surface plasmon by the surface plasmon excitation layer on the detection surface in the detection groove 73, and high sensitivity. Shows that the sample was detected.

DESCRIPTION OF SYMBOLS 1,100 Target substance detection plate 2,102 Plate main body 3,103a, 103b, 103c Flow path 4,104 Storage part 5,105 Analyte liquid storage part 4 ', 5', 107 Waste liquid storage part 6,13,23, 33, 43, 53, 63, 73, 109 Detection groove 7, 12, 22, 32, 42, 52, 62, 72 Transparent substrate part 8, 11, 21, 31, 41, 51, 61, 71, 108 Detection chip DESCRIPTION OF SYMBOLS 9 Cover part 10 Light source 14 Electric field enhancement layer 15 Space | interval 67,77,206,405 Photo detector 68 Wavelength filter 74a Chrome thin film 74b Gold thin film 74c Transparent dielectric layer 106 Cleaning liquid storage part 200 SPR sensor 201, 401a Transparent substrate 202 Metal thin film Layers 203 and 402 Optical prisms 204 and 403 Light sources 205 and 404 Polarizing plates 210A and 410A Incident light 210 , 410B reflected light 400 optical waveguide mode sensor 401 detecting plate 401b thin layer 401c optical waveguide layer A direction R side L light k fluorescence θ incident angle φ base angle

Claims (17)

Translucent formed with one or a plurality of concave accommodating parts for accommodating a detection chip for detecting a target substance, and a flow path for supplying a sample liquid for verifying the presence of the target substance in the accommodating part The plate body,
The detection chip housed in the housing portion,
The detection chip is formed on a plate-like transparent base portion that transmits light and one surface of the transparent base portion, and is connected to the flow path to introduce the analyte liquid along the flow path direction. And a detection groove
The detection groove has at least a part of an inclined surface inclined with a single gradient with respect to the one surface in a cross-sectional view, and is formed in any one of a V-shaped cross section, a trapezoidal cross section, and a polygonal cross section. is on the groove inner surface of the formed at least electric field enhancement layer is disposed, said detection groove part or the whole of the outermost surface in contact with the test solution is the detection surface of the target substance Rutotomoni, 1 A plurality of the detection chips are formed in parallel,
Wherein a spacing between grooves of the detection groove, target substance detection plate characterized that you have been first light-shielding portion is formed at a portion forming the spacing between the grooves adjacent to each other.   The target substance detection plate according to claim 1, wherein the plate body is made of a disk-shaped member. The plate body is made of a disk-shaped member, and with respect to the center of the plate, the specimen liquid storage section that stores the specimen liquid disposed at a position closer to the storage section, the cleaning liquid storage section that stores the cleaning liquid, and the storage A waste liquid storage section that stores the waste liquid composed of the analyte liquid and the cleaning liquid disposed at a position farther from the section,
2. The sample liquid storage unit, the cleaning liquid storage unit, and the waste liquid storage unit are respectively connected to the storage unit through a flow path for supplying the sample liquid, the cleaning liquid, and the waste liquid. 3. The target substance detection plate according to any one of 2 above.   The target substance detection plate according to any one of claims 1 to 3, wherein a surface opposite to the surface on which the detection groove of the transparent base portion is formed is formed flat.   The target substance detection plate according to claim 1, wherein the detection groove has a trapezoidal shape in a cross-sectional view. The target substance detection plate according to claim 5, wherein a second light-shielding portion is formed on the bottom surface of the detection groove. The target substance detection plate according to any one of claims 1 to 6, wherein the electric field enhancement layer is formed by arranging a surface plasmon excitation layer that expresses surface plasmon resonance on the groove. The target substance detection plate according to claim 7, wherein a material for forming the surface plasmon excitation layer includes at least one of gold, silver, copper, platinum, and aluminum. The target substance detection plate according to claim 7, wherein the surface of the surface plasmon excitation layer is coated with a transparent dielectric. The electric field enhancement layer is formed by arranging a thin film layer formed of a metal material or a semiconductor material and an optical waveguide layer formed of a transparent material in this order on the groove. Target substance detection plate. The target substance detection plate according to claim 10, wherein the metal material includes at least one of gold, silver, copper, platinum, and aluminum. The target substance detection plate according to claim 10, wherein the semiconductor material is silicon. The target substance detection plate according to claim 10, wherein the optical waveguide layer is formed of silica glass. The target substance detection plate according to claim 1, wherein a surface treatment for capturing the target substance is performed on the detection surface. The target substance detection plate according to any one of claims 1 to 14,
  A light irradiation means for irradiating the electric field enhancement layer with light from the surface opposite to the surface on which the detection groove of the detection chip is formed;
  A light detection means for detecting fluorescence emitted from a target substance in a sample liquid existing in the detection groove or a fluorescent substance for labeling the target substance based on the light irradiation;
  The target substance detection apparatus characterized by having. The target substance detection apparatus according to claim 15, wherein the light irradiation means includes a light source and a polarizing plate that polarizes light emitted from the light source into linearly polarized light. A target substance detection method for detecting a target substance using the target substance detection apparatus according to any one of claims 15 to 16,
  A sample liquid introduction step of sending the sample liquid into the flow path of the target substance detection plate and introducing the sample liquid into the detection groove of the detection chip;
  A light irradiation step of irradiating the electric field enhancement layer with light from the surface opposite to the surface on which the detection groove of the detection chip is formed;
  A light detection step of detecting fluorescence emitted from a target substance in a sample liquid present in the detection groove or a fluorescent substance for labeling the target substance based on the irradiation of the light;
  A target substance detection method comprising: