JP2006317426A - Substrate for analysis and analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substrate for analysis and an analyzer for enhancing detection sensitivity by suppressing the effect of background noise light on the detection sensitivity. <P>SOLUTION: This substrate 91 equipped with a cell 95 for therein holding a specimen solution is provided. A light shield layer 92 is provided on a surface of the substrate 91 provided with the cell 95. A cover layer 94 is provided on a surface of the substrate 91 on its side where the cell 95 is formed, the cover layer 94 covering the cell 95 so that a liquid is held in the cell 95. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an analysis substrate for optical analysis of a sample solution and an analysis apparatus for performing analysis using the same.

  In recent years, techniques for analyzing biological samples such as DNA, RNA, and proteins have been remarkably developed. Examples of these detection methods include a fluorescence method, an absorption method, a chemiluminescence method, and a radioisotope method. Among the above detection methods, a fluorescence method having high detection sensitivity and easy handling is used by many researchers.

  However, even in the fluorescence method, there is background light due to slight mixing of excitation light. Therefore, in order to detect fluorescence from the sample with higher sensitivity, a means for removing the background noise is further required.

  For example, Non-Patent Document 1 discloses a method of reducing background noise by providing pinholes and microlenses on both surfaces of a chip and spatially separating the optical path of incident light and fluorescence from a sample. Yes. FIG. 10 is a schematic cross-sectional view showing the state of the fluorescence detection method using this chip.

  In the fluorescence detection method, as shown in FIG. 10, a microchannel 81 through which a sample passes is provided inside a substrate-like chip 80, and the excitation light incident side surface of the chip 80, and the detection side surface corresponding to the opposite side Are provided with pinholes 82a and 82b and minute lenses 83a and 83b, respectively. The lenses 83a and 83b are disposed in the pinholes 82a and 82b, respectively.

  The incident light 85 uses parallel light that is incident on the surface of the chip 80 from an angle of 45 degrees, and the incident light 85 is condensed at one point on the microchannel 81 by the microlens 83a on the incident-side surface. The Thus, only the fluorescence 86 passing through the microlens 83b in the pinhole 82b on the detection side surface among the fluorescence emitted from the sample flowing in the microchannel 81 is detected by the photodetector.

At this time, the pinhole 82b passes a part of the sample fluorescence 86 to the detection side, and the transmitted light 87 as a result of the incident light 85 passing through the microchannel 81 does not pass to the detection side. Therefore, in the fluorescence detection method, the background light due to the transmitted light can be reduced with respect to the fluorescence 86 to be detected.
Jean-Christophe Roulet et.al., “Integration of Micro-Optical Systems for Fluorescence Detection in μTAS Applications”, Micro Total Analysis Systems 2000, pp.163

  However, in the above conventional method, since there is a distance between the microchannel 81 and the pinholes 82a and 82b, autofluorescence is emitted from the substrates 84a and 84b while the excitation lights 85 and 87 pass through the substrates 84a and 84b. Arise. Since the amount of autofluorescence is proportional to the distance it passes, the amount of autofluorescence increases with the conventional method described above. Similarly, autofluorescence is generated from the microlenses 83a and 83b. Since the autofluorescence is detected through the pinhole 82b in the same manner as the fluorescence 86 from the sample, it cannot be spatially separated from each other. In addition, autofluorescence has a wavelength range longer than that of incident light 85, which is excitation light, and is generally in the same wavelength range as fluorescence 86 from the sample, and cannot be separated by an optical filter. That is, there is a problem that the fluorescence 86 of the sample is mixed with autofluorescence from the microlenses 83a and 83b and the substrates 84a and 84b and becomes background light.

  Further, before the incident light 85 reaches the microchannel 81 through which the sample passes, the incident light is scattered at the end of the lens 83a, and the autofluorescence excited by the incident light as described above when passing through the inside of the lens 83a. Also emits. These autofluorescence and scattered light similarly become another background light that lowers the sensitivity of the fluorescence to be detected.

  That is, in the above conventional method, scattered light and self-emission emitted from the incident side lens 83a are collected by the detection side lens 83b, and a part thereof is guided to the detection side together with the fluorescence 86 from the sample. Accordingly, it is impossible to completely separate the scattered light or self-emission emitted from the incident side lens 83a and the fluorescence 86 from the sample spatially. That is, there is a problem in that the scattered light from the lens 83a or self-emission is mixed in the fluorescence 86 of the sample, and becomes background light, resulting in a decrease in detection sensitivity.

  Therefore, in order to solve this problem, it is indispensable to construct an optical system that suppresses mixing of the above-described autofluorescence / scattered light.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide an analysis substrate for detecting an optical change from a sample with high sensitivity and an analysis apparatus using the same.

  In order to solve the above problems, an analysis substrate according to the present invention is an analysis substrate capable of optical analysis of a sample solution. The analysis substrate of the present invention includes a substrate having a cell for holding a sample solution, The light-shielding layer is formed, and a cover layer is provided on the surface of the substrate on the cell formation side and covers the cell so that the liquid is maintained in the cell.

  According to said structure, a light shielding layer is pinched | interposed by the board | substrate surface in which the cell of a board | substrate exists, and a cover layer. In this structure, when incident light is incident from either the substrate or the cover layer through the condensing lens, the autofluorescence / scattered light of the substrate or cover layer and the autofluorescence / scattered light of the condensing lens are incident light. Compared to the above, the strength is weak and it is easy to be blocked by the light shielding layer, so that it does not reach the detection side opposite to the substrate.

  Thus, in the above configuration, weak autofluorescence / scattered light from the condensing lens, the substrate or the cover layer can be prevented from being observed on the detection side, whereas the incident light has high light intensity. Can be transmitted through the light-shielding layer while being reduced.

  The transmitted light can sufficiently analyze the sample in the cell. For example, the transmitted light can have sufficient intensity to excite fluorescent molecules with respect to the sample. In other words, according to the above configuration, the light shielding layer shields the autofluorescence and scattered light from the substrate or cover layer and the condensing lens, and allows the incident light to pass through while maintaining the intensity to excite the fluorescent molecules. it can.

  Therefore, in the above configuration, only the incident light is irradiated to the cell, and only the light emitted from the sample (emission from the sample), for example, fluorescence, generated in the cell reaches the detection side. Thus, it is possible to detect only light emission of the sample, for example, fluorescence with high sensitivity.

  In the analysis substrate, the light shielding layer may include an optical opening. According to the above configuration, since the collected incident light can pass through the opening without the light shielding layer in the analysis substrate, the sample held in the cell is irradiated without reducing the incident light, and the sample is efficiently It can be excited to emit light. On the other hand, the autofluorescence and scattered light of the condensing lens and the autofluorescence and scattered light of the incident side substrate or cover layer are divergent light, and most of them do not pass through the opening and are blocked by the light shielding layer. , Reaching the detection side is reduced.

  Therefore, in the above configuration, it is possible to suppress the autofluorescence and scattered light of the analysis substrate from being mixed into the light emission from the sample, and it is possible to detect only the light emission such as the fluorescence of the sample with high sensitivity. Note that the thickness of the light shielding layer can be increased in order to more completely shield autofluorescence and scattered light. At this time, since the incident light passes through the opening without being blocked by the light shielding layer, it is possible to sufficiently cut only the autofluorescence and the scattered light without reducing the incident light.

  The analysis substrate may include a protective layer that covers the light shielding layer.

  According to said structure, when the light shielding layer of the said board | substrate consists of an electroconductive substance and it is going to perform electrophoresis in the said cell, when the said protective layer is an insulator (dielectric material), the said protective layer Plays a big role. If the light shielding layer is made of a conductive material, there is a conductor along the cell, so that the conductor contacts the sample and a sufficient electric field cannot be applied in the cell, or a false electrode is formed in the cell. Inconveniences such as this occur, and good electrophoresis becomes difficult.

  Therefore, by covering the light shielding layer with a protective layer made of an insulator (dielectric material), the conductor does not come into contact with the sample, and the electric field in the cell becomes uniform in the analysis substrate, enabling good analysis.

  In order to solve the above-mentioned problems, another analysis substrate of the present invention is an analysis substrate capable of optical analysis of a sample solution. The analysis substrate is provided on a substrate having a cell for holding the sample solution and on the substrate surface. A light-shielding layer made of a reflective film, a cover layer that is provided on the surface of the substrate on the cell formation side and covers the cell so that liquid is maintained in the cell, and is formed on the substrate. Guiding means for guiding the incident light for optical analysis is provided at the position of the cell.

  According to said structure, incident light can be correctly guide | induced to the said cell by making incident means follow the guide means on the said board | substrate using an optical pick-up, for example.

  In the analysis substrate, the substrate on which the cells are engraved may be a light incident side substrate, and the cover layer may be a light emitting side substrate.

  In the above configuration, since the cell is formed on the incident-side substrate, the cover layer on the detection side can be made thinner than the incident-side substrate, and the autofluorescence and scattered light emitted from the cover layer can be reduced. The amount can be reduced.

  The analyzer according to the present invention includes a light source for making incident light incident on any of the above-described analysis substrates, and an opposite side of the light source across the analysis substrate. And a photodetector arranged at a position off the optical path.

  According to said structure, since the photodetector provided in the said analyzer is arrange | positioned in the position remove | deviated from the optical path of the said incident light, it suppresses the background noise light by direct incidence light entering low. Can do.

  Another analysis apparatus of the present invention includes a light source for making incident light incident on the analysis substrate having the guide means, and an optical pickup that detects reflected light from the analysis substrate; Moving means for moving the optical pickup in a direction along the guide means; and tracking means for controlling the optical pickup so that the incident light follows the guide means based on a detection signal from the optical pickup. It is characterized by that.

  According to the above configuration, the reflected light from the analysis substrate is read by the optical pickup, so that the incident light can be focused at an accurate position on the analysis substrate, and this state is also maintained on the cell. By doing so, it is possible to accurately irradiate the sample in the cell with the incident light and stabilize the optical analysis on the sample.

  In the above-described analyzer, a pinhole may be provided between the photodetector and the analysis substrate.

  According to the above configuration, since autofluorescence and scattered light emitted from the detection-side substrate is divergent light, by using a confocal mechanism using a pinhole, self-emission or scattered light is mixed into the photodetector. It is possible to detect luminescence such as fluorescence from the sample to be measured. In other words, it is possible to spatially separate the light emission from the sample and the autofluorescence and scattered light of the detection side substrate, thereby enabling highly sensitive emission analysis.

  As described above, the analysis substrate of the present invention is an analysis substrate capable of optical analysis of a sample solution, and is formed on a substrate provided with a cell for holding the sample solution and a substrate surface provided with the cell. The light-shielding layer, and a cover layer that is provided on the surface of the substrate on the cell formation side and covers the cell so that the liquid is maintained in the cell.

  According to said structure, a light shielding layer is pinched | interposed by the board | substrate surface in which the cell of a board | substrate exists, and a cover layer. In this structure, when incident light is incident from one of the substrate and the cover layer, for example, through a condensing lens, the autofluorescence / scattered light of the substrate or cover layer and the autofluorescence / scattered light of the condensing lens are Since the intensity is weaker than incident light and is blocked by the light-shielding layer, it is possible to suppress reaching the detection side opposite to the substrate.

  Thereby, in the said structure, while the self-fluorescence of a condensing lens, a board | substrate, or a cover layer can be prevented from being observed on the detection side, incident light, especially the incident light which condensed, has the high light intensity. Therefore, the light shielding layer can be transmitted while the light intensity is reduced.

  The transmitted light enables optical analysis of the sample in the cell. For example, the transmitted light can have sufficient intensity to excite fluorescent molecules with respect to the sample. That is, according to the above configuration, the light shielding layer shields the autofluorescence of the substrate or the cover layer or the condensing lens, and allows incident light to pass therethrough.

  Therefore, in the above configuration, only the incident light is irradiated on the cell, and only the light emitted from the sample (emission from the sample), for example, fluorescence, generated in the cell reaches the detection side. It is possible to detect the emission of the sample, for example, only the fluorescence with high sensitivity.

  Further, the analyzer of the present invention is a light source for making incident light incident on the analysis substrate, a position on the opposite side of the light source across the analysis substrate, and a position off the optical path of the incident light And a photodetector arranged in the.

  According to said structure, since the photodetector provided in the said analyzer is arrange | positioned in the position remove | deviated from the optical path of the said incident light, the background noise light by incident light can be suppressed low.

  In the analysis substrate of the present invention, when a cell near the sample is irradiated with incident light, autofluorescence / scattered light generated when the incident light passes through the substrate can be separated from light emission from the sample. In general, the shorter the wavelength of incident light, the larger the autofluorescence, and the smaller the wavelength of incident light, the smaller the autofluorescence. In the following examples, the case where the influence of autofluorescence is large and particularly large compared to scattered light will be described.

  Example 1 in Embodiment 1 of the present invention will be described below with reference to FIGS. 1 and 2. There are various types of cells that hold the sample solution, such as a channel-like one used for electrophoresis or chromatography, and a micro-cell-like one used for DNA hybridization. In the following examples, a channel will be described as an example of a cell that holds a sample solution.

  FIG. 1 is a longitudinal sectional view showing a main part of an analysis substrate 90 and an analysis incident light 98 incident on the analysis substrate 90 in the embodiment of the present invention. The cross section in the figure is a plane perpendicular to the direction in which the solution such as the sample flows in the flow channel 95.

  The analysis substrate 90 includes a substrate 91, a light shielding layer 92, an adhesive layer 93, and a cover layer 94. The substrate 91 is made of plastic or glass and has a flow path 95 through which the sample solution passes on one end surface in the thickness direction of the substrate 91. The depth of the flow path 95 is several μm to several hundred μm, and the width is several tens μm to several hundred μm. The light shielding layer 92 is laminated on one end surface of the cover layer 94 in the thickness direction. Therefore, it can be said that the light shielding layer 92 is formed on the cover layer 94.

  When a thin metal film having a thickness of 10 nm to 50 nm, such as aluminum or silver, is used as the light shielding layer 92, for example, a film that does not transmit at least the autofluorescence 97 emitted from the cover layer 94 from the incident side to the detection side, the light shielding layer 92 is It can be easily obtained as a reflective film made of a metal thin film. In addition, as the light shielding layer 92, a reflective film made of a well-known dielectric multilayer film may be used instead of the reflective film of the metal thin film, but the film thickness is about the wavelength or more. , Thicker than the metal thin film.

  The surface of the substrate 91 where the flow path 95 is provided and the surface of the cover layer 94 where the light shielding layer 92 is provided are bonded via an adhesive layer 93. For example, a UV curable resin is used for the adhesive layer 93, and the thickness is several μm to several tens of μm. The substrate 91 and the cover layer 94 are made of, for example, transparent plastic or transparent glass having light transmittance, and the thickness is, for example, 0.6 mm.

  In addition, the cover layer 94 preferably has the same size and shape as the substrate 91, but it is sufficient that the cover layer 94 has a shape that covers the flow path so that at least the sample solution flowing in the flow path 95 does not leak out of the flow path. The channel 95 is filled with the sample solution, but if the metal thin film of the light shielding layer 92 contacts this sample, there is a risk of corrosion. The adhesive layer 93 also serves to protect the light shielding layer 92 from the sample solution and prevent corrosion.

Incident light 98 is incident from the cover layer 94 side of the analysis substrate 90. The photodetector for detecting light from the sample is disposed on the substrate 91 side of the analysis substrate 90, and the incident surface and the detection surface are opposite to each other with the analysis substrate 90 interposed therebetween.
According to the above configuration, the incident light 98 incident from the cover layer 94 side is collected by the incident lens 100 onto the light shielding layer 92 below the channel 95. At this time, the incident light 98 condensed and incident on the light shielding layer 92 portion has a high light intensity on the portion due to the concentration on the portion. Therefore, the incident light 98 is incident on the central portion of the condensing spot. The light is transmitted while the light intensity is reduced. Therefore, the transmitted light 99 from the incident light 98 reaches the flow path 95. As a result, the fluorescent sample held in the flow channel 95 is excited and emits fluorescence 96. This fluorescence 96 becomes divergent light, and at least a part of them reaches a photodetector (not shown) and is detected. Note that the optical path from the air layer to the cover layer 94, the flow path 95, and the substrate 91 is drawn with the refraction at the interface of each part omitted for convenience of explanation.

  On the other hand, the incident side lens 100 and the cover layer 94 in the analysis substrate 90 emit weak autofluorescence 97 when the incident light 98 passes through. The autofluorescence 97 is diverging light, and is mostly reduced by the light shielding layer 92 in the same manner as the incident light. Accordingly, the weak autofluorescence 97 is further weakened and is almost shielded from light so that it does not reach the photodetector. That is, incident light 98 can pass through the light shielding layer 92 and irradiate the flow path 95, but the autofluorescence 97 is shielded by the light shielding layer 92. That is, the light reaching the detection side of the analysis substrate 90 is only the transmitted light 99 and the fluorescence 96 from the sample generated in the flow channel 95.

  Furthermore, as will be described later, if a photodetector is disposed outside the optical path of the transmitted light 99, the background noise due to the transmitted light 99 is not detected by the photodetector, and only the fluorescence 96 of the sample is obtained. It can be detected with high sensitivity.

  The light shielding layer 92 is usually a metal thin film, but a layer having a filter function of cutting the wavelength of the autofluorescence 97 and transmitting the wavelength of the incident light 98 may be used. In this structure, the incident light 98 can not only enter the flow path 95 without being reduced by the light shielding layer 92 but also prevent leakage of the autofluorescence 97 to the detection side.

  A dielectric multilayer film is an example of a film having such a filter function. In general, dielectric multilayer films have disadvantages such as higher manufacturing costs and thicker films than metal thin films, but it is possible to increase the fluorescence intensity from the sample by using this. .

  FIG. 2 is a front view showing the main part of the optical system in the light detection device 110 provided in the analysis device according to the present invention, which detects fluorescence of the sample from the analysis substrate 90 in FIG.

  The incident light 98 for analysis emitted from the light source 117 is collected by the lens 100 and enters the flow path 95. The fluorescence 96 from the sample flowing through the flow path 95 is condensed by the lens 111 to become parallel light, and is picked up by the optical filter 112. The optical filter 112 has a property of significantly reducing at least light in the wavelength band of excitation light and sufficiently transmitting the wavelength band of fluorescence from the sample. The parallel light that has passed through the optical filter 112 is collected by the lens 113, passes through the pinhole 114 at the focal position of the lens 113, and is then converted into an electrical signal by the photodetector 115.

  The light path collected from the sample by this optical system is shown as 116. This optical system is disposed outside the optical path of the transmitted light 99. The lens 111 is a so-called confocal optical system because its focal point is defined by the flow path 95.

  According to the above configuration, the light detection device 110 provided in the analyzer is disposed outside the optical path of the transmitted light 99. Therefore, since each optical component (111-113) is not irradiated to the transmitted light 99, it does not emit self light from there. If these optical components are irradiated with the transmitted light 99, the transmitted light 99 has a very strong light intensity, so that the optical component exposed to the transmitted light 99 emits autofluorescence. This self-emission may be detected after passing through the optical filter 112. Further, the transmitted light 99 itself may slightly leak from the optical filter 112 and enter the photodetector 115. These autofluorescence and transmitted light leakage light become background light, but can be suppressed by arranging the photodetection device 110 outside the optical path of the transmitted light 99 as described above.

  In addition, a so-called confocal optical system that allows only light passing through the focal point of the lens 113 to pass through the pinhole 114 is formed. Therefore, only fluorescence 96 from the sample to be measured can be guided to the photodetector 115. That is, even if the autofluorescence is generated from the substrate 91 by the transmitted light 99, most of the autofluorescence is blocked by the pinhole 114 in the confocal optical system and is not detected.

  However, if the light receiving surface of the photodetector 115 is very small and the pinhole 114 can also be used, the pinhole 114 can be omitted. Further, when the light receiving area of the photodetector 115 is large, the lens 111 and the lens 113 for condensing fluorescence may be omitted.

  As described above, the analysis substrate 90 of the present invention includes the substrate 91 provided with the flow path 95 for holding the sample solution, the light shielding layer 92 formed on the substrate surface provided with the flow path 95, and the substrate 91. It is provided with a cover layer 94 provided on the surface on the formation side of the flow channel 95 and covering the flow channel 95 so that the liquid in the flow channel 95 is maintained. The light shielding layer 92 is preferably formed on the surface of the substrate 91 provided with the flow path 95 so as to cover the entire surface.

  According to the above configuration, the light shielding layer 92 is sandwiched between the substrate surface where the flow path 95 of the substrate 91 exists and the cover layer 94. In this structure, when incident light is incident on the cover layer 94 through the condensing lens 100, the autofluorescence 97 of the cover layer 94 and the autofluorescence 97 of the lens 100 have low light intensity, and are easily formed by the light shielding layer 92. Therefore, the detection side opposite to the analysis substrate 90 is not reached.

  This prevents the lens 100 and the autofluorescence 97 of the cover layer 94 from being observed on the detection side. On the other hand, the condensed incident light 98 has a high light intensity, so that the central portion of the condensed spot transmits the light shielding layer 92 while the incident light 98 is reduced by the light shielding layer 92. The transmitted light 99 has sufficient intensity to excite the fluorescent sample in the flow channel 95. In other words, the light shielding layer 92 shields the cover layer 94 and the autofluorescence 97 of the lens 100, and transmits the incident light 98 to excite the sample.

  Accordingly, only the transmitted light 99 from the incident light 98 is irradiated to the flow path 95, and only the sample fluorescence 96 generated in the flow path 95 reaches the detection side. Therefore, mixing of the autofluorescence 97 described above can be suppressed, and only the fluorescence 96 of the sample can be detected with high sensitivity.

  The analysis apparatus of the present invention is located on the opposite side of the light source 117 across the analysis substrate 90, the light source 117 that makes incident light incident on the analysis substrate 90, and the analysis substrate 90, and transmits light. And a photo detector 115 arranged in other than 99 optical paths. According to this, since the photodetector 115 provided in the analyzer is arranged outside the optical path of the transmitted light 99 from the incident light 98, the transmitted light 99 is directly incident on the photodetector 115. However, the background noise light caused by the transmitted light 99 can be kept low.

  Therefore, according to the present invention, it is possible to provide an analysis substrate and an analysis apparatus for detecting an optical change from a sample or a reagent with high sensitivity. The method of electrophoresis in the flow path 95 of the analysis substrate 90 will be described in detail with reference to FIG.

  A second embodiment as another embodiment of the present invention will be described below with reference to FIGS. In the following embodiment, a description will be given by taking a flow path in electrophoresis as an example of a cell for holding a sample solution.

  FIG. 3 is a longitudinal sectional view showing a main part of the analysis substrate 1 in this embodiment and a state in which the incident light 12 for analysis is incident on the analysis substrate 1. The cross section in the figure is a plane perpendicular to the direction in which a solution such as a sample flows in the flow path 8 and is a plane obtained by cutting the analysis substrate 1 in a direction along the guide groove 5 (guide means).

  The analysis substrate 1 includes a substrate 2, a light shielding layer 3, a protective layer 4, a cover layer 7, and an adhesive layer 6. The substrate 2 is made of, for example, transparent plastic or transparent glass having light transmittance, and includes a flow path 8 through which the sample solution passes and a guide groove 9 at other locations. The light shielding layer 3 is laminated on the substrate 2 which is a place other than the flow path 8 on the surface of the substrate 2 where the flow path 8 is formed.

  As the light shielding layer 3, a similar reflective film described in the first embodiment may be used. The portion of the substrate 2 where the light shielding layer 3 is not laminated (that is, the portion facing the flow path 8) becomes an opening 3a through which light can pass. The width of the opening 3a is several tens of μm to several mm. In order to sufficiently shield the light, it is preferably several tens to several hundreds of μm. The protective layer 4 is laminated so as to cover the light shielding layer 3, and the adhesive layer 6 is for adhering the cover layer 7 on the substrate 2.

  For example, on the substrate 2 having a thickness of 0.6 mm, a groove-like flow path 8 molded in a groove shape is formed on one surface, and guide grooves 9 are formed on both sides of the flow path 8. The guide groove 9 is cut in a direction perpendicular to the flow path 8, which is the structure of the analysis substrate shown in Japanese Patent Application No. 2004-242978 (name of invention: analysis substrate and analysis apparatus). It is the same. The depth of the flow path 8 is several μm to several hundred μm, and the width is several tens μm to several hundred μm.

  A light shielding layer 3 made of a reflective film is formed on a portion of the surface of the substrate 2 where the flow channel 8 is located except for the flow channel 8 (portion of the opening 3a). As a result, the light shielding layer 3 takes the form of covering the guide grooves 9 carved in the substrate 2. As will be described later, the light shielding layer 3 is made of a film that does not transmit at least light having the wavelength of incident light and autofluorescence emitted from the cover layer 7 to the detection side.

  By the way, in the case of electrophoresis, if a metal film is used for the light shielding layer 3, the electric field tends to be zero (constant potential) in the vicinity of the metal film, and the electrophoresis in the flow path 8 is likely to be hindered. Therefore, in the case of electrophoresis, a sufficient interval is required between the light shielding film 3 and the flow path 8. Further, instead of the metal film, a reflective film made of a dielectric multilayer film may be used. In this way, by using a reflective film made of a metal film or a dielectric multilayer film, the light shielding layer 3 also functions as a reflective film necessary for reading a guide groove 5 described later.

  The protective layer 4 is formed so as to cover the light shielding layer 3. A guide groove 9 is carved in a concavo-convex shape on the substrate 2, and the light shielding layer 3 and the protective layer 4 are formed with a uniform thickness thereon. Eventually, on the protective layer 4, the guide groove 5 reflecting the same uneven shape as the guide groove 9 is formed.

  The protective layer 4 is bonded to the cover layer 7 via the adhesive layer 6. For the adhesive layer 6, for example, a UV curable resin is used, and the thickness is several μm to several tens of μm. The cover layer 7 is made of, for example, transparent plastic or transparent glass having light transmissivity, and has a thickness of, for example, 0.6 mm.

  The cover layer 7 is preferably formed to have the same size and shape as the substrate 2, but it is sufficient that the cover layer 7 has a shape covering the flow path 8 so that at least the sample solution flowing through the flow path 8 does not leak. Thereby, the flow path 8 is sealed and it is possible to prevent the reagent solution flowing through the flow path 8 from leaking to other places.

  Incident light 12 is incident from the cover layer 7 side of the analysis substrate 1. The photodetector for detecting light from the sample is disposed on the substrate 2 side of the analysis substrate 1, and the incident surface and the detection surface are opposite to each other with the analysis substrate 1 in between.

  According to the above configuration, the incident light 12 incident from the cover layer 7 side in the analysis substrate 1 is collected by the incident-side condensing lens 14, and the flow without the light shielding layer 3 in the analysis substrate 1 is obtained. Road 8 can be illuminated. At this time, the sample in the flow path 8 emits fluorescence 10 by the incident light 12. The fluorescence 10 becomes divergent light and reaches a photodetector (not shown) and is detected. On the other hand, after the incident light 12 passes through the flow path 8, it goes out of the substrate 2 through the optical path of the transmitted light 13. Note that the optical path from the air layer to the cover layer 7, the flow path 8, and the substrate 2 is drawn with the refraction at the interface of each part omitted for convenience of explanation.

  The incident side lens 14 and the cover layer 7 emit weak autofluorescence 11 when the incident light 12 passes through. The autofluorescence 11 is divergent light, and is mostly shielded by the light shielding layer 3 and does not reach the photodetector. Therefore, the light existing on the detection side is only the transmitted light 13 and the fluorescence 10 of the sample generated in the flow path 8 as described above.

  Further, as will be described later, if a photodetector is disposed outside the optical path of the transmitted light 13, the background noise due to the transmitted light 13 is not detected, and only the fluorescence 10 of the sample is detected, and can be detected with high sensitivity. .

  The light shielding layer 3 is preferably a metal thin film reflective film. When incident light is focused on the guide groove 5 on the reflective film, diffracted light from the guide groove 5 is reflected to an optical pickup described later (not shown). When the reflected light is used for servo control with an optical pickup, the incident light 12 is guided on the guide groove 5 and can be accurately guided to a desired position along the flow path 8.

  However, if a metal film is used for the light shielding layer 3, the metal film is exposed to the flow path 8, so that a false electrode can be formed on the analysis substrate 1. Therefore, in this case, the generation of a false electrode can be prevented by covering with the protective layer 4 of an insulator.

  The reason is as follows. When the light shielding layer 3 is a conductive substance, a conductor exists along the flow path 8. Even if a voltage for electrophoresis is applied thereto, an electric field is hardly generated in the flow path because of this conductor. That is, good electrophoresis becomes difficult. Therefore, by covering the conductive light shielding layer 3 with the protective layer 4 made of an insulator (dielectric material), electrophoresis can be performed without causing the sample in the flow channel to contact the conductive light shielding layer 3.

  However, as described above, when a metal film is used, it is difficult to apply an electric field for electrophoresis. Therefore, it is necessary to take a distance between the flow path and the metal film as much as possible. Therefore, the area of the opening 3a in the light shielding layer 3 is widened, but this is determined by the balance with the electric field strength. When a dielectric multilayer film is used instead of the metal film, the opening 3a is not widened, and the protective layer 4 can be omitted.

  The adhesive layer 6 may be formed so as to completely cover the light shielding layer 3 so that the light shielding layer 3 is not exposed to the flow path 8. In this case, since the adhesive layer 6 can also function as the protective layer 4, the protective layer 4 can be omitted.

  The apparatus for detecting the fluorescence of the sample from the analysis substrate 1 is the same as the light detection apparatus 110 of the first embodiment, and a detailed description thereof will be omitted. The difference from the first embodiment is that an optical pickup is used instead of the light source 117. As a result, the guide groove 5 of the analysis substrate 1 can be read as will be described later, and the incident light 12 can be incident on a desired position.

  FIG. 4 shows an example in which the configuration of the main part of the analysis substrate 1 described above is applied to the disk-shaped substrate 30 and analysis is performed using electrophoresis. The disk-shaped substrate 30 for analysis can perform analysis using electrophoresis. In the figure, a center hole 31 for fixing the disk-shaped substrate 30 to the center of the turntable of the analyzer, four analysis chips 32 for analyzing the sample, and each liquid reservoir / injection port 34a constituting this are shown. To 34d, the first and second migration paths 35a and 35b, the guide groove 5, and the address information recording section 33 are shown. Further, the filled portion indicates a portion where the light shielding layer 3 is provided. Therefore, only the portions facing the first migration path 35a, the second migration path 35b, and the liquid reservoir / injection ports 34a to 34d are provided. Is not formed. In other words, the first migration path 35a, the second migration path 35b, and the liquid reservoir / injection ports 34a to 34d are formed with slits 36 that are openings 3a. A cross-sectional view of the portion where the slit 36 is formed is as shown in FIG. 1 or FIG.

  In the analysis chip 32 for electrophoresis, a first migration path 35a and a second migration path 35b corresponding to the flow path 8 are formed in a cross shape orthogonal to each other. The structure of the analysis chip 32 is a commonly used structure, and is disclosed in, for example, Japanese Patent Application Laid-Open No. 2003-56003 (published March 5, 2003) together with an analysis example.

  The first migration path 35a extends in the radial direction of the disk-shaped substrate 30 for analysis, and the second migration path 35b extends perpendicular to the first migration path 35a. Each of the liquid reservoir / injection ports 34 a to 34 d is disposed at four ends of the migration path 35 so as to communicate with the migration path 35. That is, a liquid reservoir / injection port 34a is disposed at one end of the second migration path 35b, and a liquid reservoir / injection port 34c is disposed at the other end, and the liquid reservoir / injection port 34b is disposed at one end of the first migration path 35a. A liquid reservoir / injection port 34d is arranged in the part.

  In the disk-shaped substrate 30 described above, in the analysis process (electrophoresis process), the buffer / gel solution is injected from one of the liquid reservoir / injection ports 34a to 34d shown in FIG. The migration path 35b is filled so that air does not enter. Thereafter, the sample solution is injected into the liquid reservoir / injection port 34a. For example, DNA is used for the sample solution.

  This DNA needs to be fluorescently labeled by a method such as introduction of a fluorescent dye at the terminal or mixing with an intercalator type fluorescent dye. Next, the electrode supplied from the apparatus power supply is attached to each liquid reservoir / injection port as follows. First, an electrode placed in the liquid reservoir / injection port 34c is connected to a + voltage power source (several tens to several hundreds of volts), and an electrode placed in the liquid reservoir / injection port 34a is connected to the ground.

  At this time, the electrode placed in the liquid reservoir / injection port 34b and the electrode placed in the liquid reservoir / injection port 34d are kept open. As a result, a positive voltage (several tens to several hundreds of volts) is applied to the liquid reservoir / injection port 34c, zero volts is applied to the liquid reservoir / injection port 34a, and the injected sample (DNA fragment: negatively charged) is second. In the migration path 35b, the electrophoresis proceeds from the liquid reservoir / injection port 34a (-electrode) toward the liquid reservoir / injection port 34c (+ electrode).

  Next, after the migrated sample reaches the crossing point of the cross in the migration path 35, the liquid reservoir / injection ports 34a and 34c are electrically opened. Next, the electrode of the liquid reservoir / injection port 34d is connected to a + voltage power source (several tens to several kilovolts), and the electrode of the liquid reservoir / injection port 34b is connected to the ground. Therefore, the electrodes for electrophoresis are switched from the electrodes of the liquid reservoir / injection ports 34a, 34c to the electrodes of the liquid reservoir / injection ports 34b, 34d.

  As a result, a positive voltage (several tens to several kilovolts) is applied to the electrode of the liquid reservoir / injection port 34d, zero volt is applied to the electrode of the liquid reservoir / injection port 34b, and the injected sample passes through the first migration path 35a. The inside migrates from the intersection of the ten characters toward the liquid reservoir / injection port 34d. At this time, since the mobility of the sample (DNA) varies depending on the molecular weight, it can be divided into fragments for each molecular weight. Since the principle of electrophoretic analysis in such a microchannel is well known as disclosed in the above-mentioned Japanese Patent Application Laid-Open No. 2003-56003, detailed description thereof is omitted.

  The first migration path 35a is irradiated with incident light as shown in FIG. 1 or 3 which is a cross-sectional view of this portion, and the sample emits fluorescence when passing through the fluorescently labeled sample. At this time, among the autofluorescence emitted from the cover layer 7 and the lens 14 as described above, only a small amount passes through the slit 36, and leakage to the detection side can be suppressed.

  In addition, by using the light detection device shown in FIG. 2, it is possible to suppress the background noise light due to the transmitted light 13 and the autofluorescence in the substrate 2. Therefore, only the fluorescence of the sample can be detected with high sensitivity.

  In addition to the analysis chip 32, the disk-shaped substrate 30 is provided with a guide groove 5 for guiding incident light and an address information recording unit 33 in which address information is recorded. When the disk-shaped substrate 30 rotates, the incident light 12 is accessed to a desired radial position of the disk-shaped substrate 30 while reading out the radial position included in the address information. Fluorescence detection (analysis) can be performed at a desired position along.

  Further, a total of four analysis chips 32 are arranged, and those adjacent to each other in the circumferential direction are provided at an angle of 90 degrees. Therefore, between the adjacent analysis chips 32, the above-described guide groove 5 and the address information recording unit 33 are formed side by side in the circumferential direction. Incident light 12 detects the fluorescence at the desired analysis chips in the four analysis chips 32 in order to access the analysis chips 32 arranged in the circumferential direction while reading the number of the analysis chip 32 included in the address information. (Analysis) is possible.

  At this time, when the incident light 12 scans one track (guide groove 5) in the circumferential direction, these are repeated in the order of the tracking area of the guide groove 5 → the address information recording unit 33 → the tracking area of the guide groove 5 → the migration path 35. Will be scanned. FIG. 5 is a longitudinal sectional view of the analysis substrate 1 showing a state in which the analysis incident light 12 passes through the flow path 8 and moves to the tracking region of the guide groove 5 by tracking the incident light 12. FIG.

  As shown in the figure, the incident light 12 for analysis passes through the optical path and is collected in the guide groove 5. At this time, the incident light 12 is reflected by the reflective film used for the light shielding layer 3 and returns to the lens 14 through the optical path 41. At this time, since the incident light 12 is diffracted in the guide groove 5 and the distribution of the diffracted light is reflected in the reflected light, the incident light 12 can follow the guide groove 5 by an optical pickup described later.

  Therefore, if the guide groove 5 is formed so as to cross the desired detection position of the flow path 8 (but not in the flow path 8 itself), the incident light is on the extension of the guide groove 5. The desired position of the flow path 8 is traversed, and the fluorescence of the sample can be detected (analyzed) at that position.

  Further, as will be described later, since tracking control is maintained during a period of passing through the flow path 8, even if the guide groove 5 does not exist in the flow path 8 itself, the guide groove 5 ′ before passing through the flow path 8 is used. Thus, stable tracking is performed continuously to the guide groove 5 on the extension. In the above-described example, the guide groove is described as an example of the guide means. However, the present invention is not limited to this, and for example, a wobble pit may be used as the guide means.

  FIG. 6 is a block diagram showing the configuration of the main part of the incident light control apparatus 50 for the analysis substrate 1 in the present embodiment. The incident light control device 50 includes an optical pickup 51, an address reproducing circuit 52, a controller 53 (tracking means), and a servo circuit 54 (tracking means).

  In addition to the lens 14 and the actuator 51a, the optical pickup 51 has an optical system 51b composed of optical components such as a semiconductor laser, a photodetector, and a beam splitter. In the figure, the lens 14 and the actuator 51a are components constituting the optical system 51b, but are illustrated separately from the optical system 51b for the sake of clarity.

  In the incident light control device 50, incident light emitted from the optical system 51 b is collected by the lens 14 and is incident on the analysis substrate 1. This incident light is applied to the guide groove 5 or the address information recording unit 33 of the analysis substrate 1. The reflected light from the guide groove 5 or the address information recording unit 33 returns to the optical pickup 51 again and outputs a focus error signal / track error signal 55 from the optical system 51b.

  The focus error signal / track error signal 55 is fed back to the actuator 51a via the servo circuit 54 and controls the actuator 51a so that the incident light is guided to the guide groove 5 or the address information recording unit 33. The light quantity detection signal 56 is input to the address reproduction circuit 52, detects address information 57, and sends the address information 57 to the controller 53.

  The controller 53 outputs a control signal 58 to the servo circuit 54 while confirming the address information 57, and performs a process of allowing incident light to access a desired guide groove. Thereby, the incident light is moved to the radial position on the disk-shaped analysis substrate 1, and fluorescence detection can be performed at a desired position along the flow path 8.

  Further, the analysis substrate 1 is rotated, and fluorescence detection is performed with an analysis chip having a desired number. Note that the tracking state in the immediately preceding guide groove is maintained on the flow path 8 without the guide groove 5. In order to maintain the time, it is sufficient that the time crossing the flow path 8 is sufficiently shorter than the response time of the servo (proportional to the reciprocal of the servo band). Or what is necessary is just to hold | maintain a servo operation temporarily, when crossing the flow path 8 (hold operation | movement).

  By doing so, the tracking is not disturbed even if the incident light 12 crosses the flow path 8, and the sample is irradiated with the incident light 12 accurately, and then reaches the guide groove 9 again to return to the tracking operation. be able to.

  As described above, in this embodiment, as in the first embodiment, the analysis substrate 1 has the light shielding layer 3 that suppresses the autofluorescence 11 from the lens 14 and the cover layer 7, and the detection system is the optical path of the transmitted light 13. Arranged outside.

  In addition, the analysis substrate 1 of this embodiment is provided with an opening 3 a that guides the incident light 12 to the flow path 8. Thereby, since the condensed incident light 12 can pass through the opening 3a without reducing the light intensity, the fluorescent sample held in the flow path 8 can be excited efficiently.

  Further, since the width of the opening 3a is several tens of μm to several mm, most of the autofluorescence 11 of the lens 14 and the autofluorescence 11 of the cover layer 7 which are diverging light are blocked by the light shielding layer 3. Since the autofluorescence 11 passing through the part 3a is in a very small amount, it does not reach the detection side or does not affect the detection even if it reaches the detection side. Therefore, mixing of the autofluorescence 11 from the lens 14 and the cover layer 7 can be suppressed, and only the fluorescence 10 of the sample can be detected with high sensitivity.

  In addition, the thickness of the light shielding layer 3 can be increased in order to further completely shield the autofluorescence 11 from light. At this time, the autofluorescence 11 is completely shielded from light, while the incident light 12 can pass through the opening 3 a without being shielded by the light shielding layer 3.

  Therefore, in this embodiment, it is possible to provide an analysis substrate and an analysis apparatus for detecting an optical change from a sample or a reagent with high sensitivity.

  In this embodiment, the slit 36 is used as the opening 3a. However, the present invention is not limited to this, and a minute opening such as a pinhole may be used. When the slit 36 along the flow path 8 is used in the opening 3a as in the present embodiment, optical analysis at an arbitrary position of the flow path 8 is possible. In addition, when a pinhole is used as the opening 3a, autofluorescence / scattered light from the lens or cover layer reaching the detector can be reduced as compared with the case of the slit.

  Example 3 as still another embodiment of the present invention will be described below with reference to FIGS. In the following embodiment, only differences from the embodiment 2 will be described, and description of other same points will be omitted.

  FIG. 7 is a longitudinal sectional view showing a main part of the analysis substrate 60 in the embodiment of the present invention and a state where the incident light 71 for analysis is incident on the analysis substrate 60. This figure is a surface perpendicular to the direction in which a solution such as a sample flows in the flow path 67 as in FIG. 1 in the first embodiment, and a surface obtained by cutting the analysis substrate 60 in the direction along the guide groove 64. It is.

  The configuration of the analysis substrate 60 is almost the same as that of the analysis substrate 1 of FIG. 3, and includes a disk-shaped substrate 61, a light shielding layer 62, a protective layer 63, a cover layer 66, and an adhesive layer 65. The disk-shaped substrate 61 is made of, for example, transparent plastic or transparent glass having light transmittance, and includes a flow path 67 through which the sample solution passes and a guide groove 68 at other locations. The light shielding layer 62 is laminated at a place other than the channel 67 on the surface of the disk-shaped substrate 61 where the channel 67 is present, and is preferably a reflective film. The portion where the light shielding layer 62 is not laminated becomes an opening 62a through which light can pass. The protective layer 63 is laminated so as to cover the light shielding layer 62, and the adhesive layer 65 bonds the disk-shaped substrate 61 and the cover layer 66.

  There are two differences from the analysis substrate 1 in Example 2 described above. The first point is that the opening 62a is asymmetric with respect to the flow path 67 and is wide on the detector side in the surface direction of the disk-shaped substrate 61, and is narrower on the opposite side than the detector side. It is a point. The second point is that the disk-shaped substrate 61 is thickened, while the cover layer 66 is thinned.

  The disk-shaped substrate 61 having a thickness of 1.2 mm has a channel 67 formed in a groove shape on one surface (surface on the detector side), and guide grooves 68 are formed on both sides of the channel 67. ing. The guide groove 68 is cut in a direction perpendicular to the channel 67. The depth of the flow path 67 is several μm to several hundred μm, and the width is several tens μm to several hundred μm.

  A light shielding layer 62 made of a reflective film is formed on a portion of the disk-like substrate 61 other than the flow channel on the surface having the flow channel 67. Thereby, a reflective film is formed in the guide groove 68. The opening 62a is provided so as to be wider on the detector side than the analysis substrate 1 of the second embodiment. The property of the light shielding layer 62 is the same as that of the light shielding layer 3 and is made of a film that does not transmit at least the wavelength of the incident light 71 and the autofluorescence 70 emitted from the disk-shaped substrate 61 to the detector side. For example, as described above, a dielectric reflection film or a metal thin film such as aluminum or silver is used.

  The protective layer 63 is formed so as to cover the light shielding layer 62. A guide groove 68 is carved in a concavo-convex shape on the disk-shaped substrate 61, and the light shielding layer 62 and the protective layer 63 are formed with a uniform thickness thereon. Eventually, on the protective layer 63, the guide groove 64 reflecting the same uneven shape as the guide groove 68 is formed.

  The protective layer 63 is bonded to the cover layer 66 through the adhesive layer 65. For example, a UV curable resin is used for the adhesive layer 65, and the thickness is several μm to several tens of μm. The cover layer 66 is made of, for example, transparent plastic or transparent glass having a light transmission property, and the thickness is set to 0.1 mm, for example. The cover layer 66 preferably has the same size and shape as the disk-shaped substrate 61 in the plane direction, but it is sufficient that the cover layer 66 has a shape that covers the channel 67 so that at least the sample solution flowing through the channel 67 does not leak. . Thereby, the flow path 67 is sealed and it is possible to prevent the reagent solution flowing through the flow path 67 from leaking to other places.

  Incident light 71 is incident from the disk-shaped substrate 61 side of the analysis substrate 60. The photodetector for detecting light from the sample is disposed on the cover layer 66 side of the analysis substrate 60, and the incident surface and the detection surface are opposite to each other across the analysis substrate 60.

  According to the above configuration, the incident light 71 incident from the disk-shaped substrate 61 side is condensed by the incident-side condensing lens 73 and irradiates the flow path 67 without the light shielding layer 62 in the analysis substrate 60. . At this time, fluorescence 69 is emitted from the sample by the incident light 71 to the sample in the flow path 67. The fluorescence 69 becomes divergent light and reaches the photodetector to be detected.

  However, in the analysis substrate 60 in the present embodiment, since most of the flow path 67 is disposed on the incident side with respect to the light shielding layer 62, when the same opening as in the second embodiment is used, The optical path is blocked by the end of the light shielding layer 62, and the amount of fluorescent light that reaches is reduced. Therefore, in order to send more fluorescence to the detector side as described above, it is desirable that the opening 62a is wide only on the detector side.

  Thereby, the fluorescence 69 from the sample emitted from the flow path 67 can be sufficiently emitted to the detector side by passing through the widened opening 62a. On the other hand, the incident light 71 passes through the flow path 67 and then exits from the cover layer 66 side through the optical path of the transmitted light 72. The condensing lens 73 and the cover layer 66 on the incident side emit weak autofluorescence 70 when the incident light 71 passes through. The autofluorescence 70 is divergent light, and is mostly shielded by the light shielding layer 62 and does not reach the photodetector.

  Therefore, the light on the detector side is only the transmitted light 72 and the fluorescence 69 of the sample generated in the channel 67 as described above. Furthermore, as will be described later, if a photodetector is arranged outside the optical path of the transmitted light 72, the transmitted light 72 is not detected by the photodetector, and only the fluorescence 69 of the sample is detected. 69 can be detected with high sensitivity.

  For the same reason as the analysis substrate 1, the analysis substrate 60 uses a metal reflective film for the light shielding layer 62. Thereby, the incident light 71 condensed at a desired position along the flow path 67 can be accurately guided in the same manner as the analysis substrate 1. Further, by covering the light shielding layer 62 with the protective layer 63 made of an insulator, good electrophoresis can be performed.

  Alternatively, the adhesive layer 65 may be formed so as to completely cover the light shielding layer 62 so that the light shielding layer 62 is not exposed to the channel 67. In this case, since the adhesive layer 65 can also function as the protective layer 63, the protective layer 63 is omitted.

  FIG. 8 is a front view showing a main part of the detection optical system of the light detection device 74 as an analysis device for detecting the fluorescence 69 of the sample from the analysis substrate 60 in FIG.

  In the detection optical system, the fluorescence 69 from the sample flowing through the flow path 67 is collected by the lens 75 to become parallel light and is picked up by the optical filter 76. This optical filter 76 has a property of significantly reducing at least light in the wavelength band of excitation light and sufficiently transmitting the wavelength band of fluorescence 69 from the sample. The parallel light that has passed through the optical filter 76 is collected by a lens 77 and converted into an electrical signal by a photodetector 78. The light path collected from the sample by this detection optical system is shown as 79. The optical system is disposed outside the optical path of the transmitted light 72 described above.

  According to the above configuration, the light detection device 74 provided in the analysis device is disposed on a path other than the optical path of the transmitted light 72. Therefore, since each optical component (75-77) is not irradiated to the transmitted light 72, it does not emit self light from there. If these optical components are irradiated to the transmitted light 72, the transmitted light 72 has a very strong light intensity, so that the optical component emits autofluorescence. This autofluorescence may be detected through the optical filter. Further, the transmitted light 72 itself may slightly leak and enter the photodetector 78. These autofluorescence and transmitted light leakage light become background light, but can be suppressed by arranging the light detection device 74 outside the optical path of the transmitted light 72 as described above.

  Further, the pinhole 114 used in the first embodiment can be deleted from the optical system in the present embodiment. This is because the thickness of the substrate 2 on the detection side in Example 1 is 0.6 mm, whereas in this embodiment, the flow path 67 is formed in the disk-shaped substrate 61 to thereby provide a cover layer on the detection side. This is because the thickness of 66 is 0.1 mm, which is smaller than the thickness of the disk-shaped substrate 61. The cover layer 66 emits autofluorescence by the transmitted light 72, but is as thin as 0.1 mm, and the amount of autofluorescence is small.

  Therefore, it is sufficiently smaller than the self-emission of the substrate 91 on the detection side in the first embodiment. For this reason, pinholes can be deleted. Note that when the fluorescence intensity of the fluorescence 69 from the sample is sufficiently high, it is not necessary to condense the lens, so that the lenses 75 and 77 may be omitted and only the optical filter 76 and the photodetector 78 may be used.

  In Examples 1, 2 and 3, details of the electrode wiring method for performing electrophoresis, a device for supplying power, a circuit for detecting fluorescence are not described, but Japanese Patent Application No. 2004-242978 Since it is the same as the method described, description is abbreviate | omitted.

  In each of the above-described Examples 2 and 3, examples of fluorescence detection in electrophoresis have been shown. However, the present invention is not limited to this, and can be applied to fluorescence detection such as hybridization and chromatography. In addition, although the analysis substrate has been shown as an example of a disk shape, the fluorescence sensitive detection method using the light shielding layer is not limited to this, and other shapes such as a card type can be used.

  A fourth embodiment as still another embodiment of the present invention will be described below with reference to FIG. In the following embodiment, only differences from the above-described embodiments will be described, and description of other identical points will be omitted.

  An analysis substrate 120 shown in FIG. 9 is an example of Example 4 according to the present invention having a shape similar to a so-called DNA chip. The analysis substrate 120 includes a cell 121 for DNA hybridization and a guide groove 122. In the cell 121, the fluorescent molecule of the hybridized DNA emits light, and this can be detected with high sensitivity using the above-described light shielding layer. Moreover, although the example of fluorescence detection was shown, it is applicable to the board | substrate and apparatus which detect light emission by excitation light sources, such as phosphorescence, with high sensitivity.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.

  In each of the above Examples 1 to 4, an example in which the wavelength of incident light is short and the autofluorescence is reduced by the light shielding layer is shown. However, the present invention is not limited to this, and it goes without saying that even when the wavelength of incident light is relatively long and the scattered light is stronger than the autofluorescence, the light shielding layer can reduce the scattered light.

  In the analysis substrate and analysis apparatus of the present invention shown in the following examples, light emitted from a sample can be separated from scattered light and autofluorescence generated when incident light is irradiated onto a condenser lens. In general, the shorter the wavelength of incident light, the greater the influence of autofluorescence. On the other hand, the longer the wavelength of incident light, the less the autofluorescence and the greater the effect of scattered light. In the following examples, the case where the influence of scattered light is greater than that of autofluorescence (incident light wavelength is 650 nm) will be described.

  Example 5 in the embodiment of the present invention will be described below with reference to FIGS. As described above, the cell holding the sample solution has various shapes such as a flow channel used for electrophoresis and chromatography, and a micro-cell shaped one used for DNA hybridization. . In the following examples, a channel will be described as an example of a cell that holds a sample solution.

  FIG. 11 is a longitudinal sectional view showing a main part of the analysis substrate 151 and a state where incident light 98 is incident in the embodiment of the present invention. The cross section in the figure is a plane perpendicular to the direction in which the solution such as the sample flows in the flow channel 95.

  The analysis substrate 151 includes a substrate 91, a cover layer 94, and a light shielding layer 92. The substrate 91 is made of a transparent plastic, has a thickness of 0.6 to 1.2 mm, and has a flow path 95 through which the sample solution passes on one end surface of the substrate 91 in the thickness direction. The depth of the flow path 95 is several μm to several hundred μm, and the width is several tens μm to several hundred μm. That is, a flow path (cell) that holds the sample solution at a distance corresponding to the thickness of the cover layer 94 from the surface of the analysis substrate 151 is incorporated. The light shielding layer 92 is laminated on one end surface of the cover layer 94 in the thickness direction. Therefore, the light shielding layer 92 is formed on the cover layer 94. The cover layer 94 may be provided with a tracking guide groove 5 described later. This will be described in Embodiment 2 with reference to FIG.

  The light shielding layer 92 is formed of a film that does not transmit at least scattered light emitted from the objective lens 100 and autofluorescence 97 from the incident side to the detection side. For example, a metal thin film having a thickness of 10 nm to 100 nm such as aluminum or silver is used. Then, the light shielding layer 92 can be easily obtained as a reflective film made of a metal thin film. This metal thin film is formed by sputtering, for example.

  The surface of the substrate 91 with the flow path 95 and the surface of the cover layer 94 without the light shielding layer 92 are easily joined by thermocompression bonding between plastics. In addition to this, for example, UV curable resin may be used, and both surfaces may be joined via an adhesive layer having a thickness of several μm to several tens of μm. The cover layer 94 is made of, for example, transparent plastic having light transmittance, and has a thickness of 0.03 to 0.6 mm.

  Since the metal thin film of the light shielding layer 92 may be oxidized when exposed to the atmosphere, it is better to form a dielectric protective layer 4 thereon. For example, the protective layer 4 has a silicon nitride thickness of 20 nm.

  Incident light 98 is incident through an opening 92 a formed in the light shielding layer 92. The width of the opening 92a is several tens of μm to 2 mm. The photodetector for detecting light from the sample is disposed on the substrate 91 side of the analysis substrate 151, and the incident surface and the detection surface are opposite to each other across the analysis substrate 151. According to the above configuration, the incident light 98 is condensed on the surface of the light shielding layer 92 at a position facing the flow path 95 by the incident lens 100 and is incident from the opening 92a.

  At this time, incident light 98 condensed and incident on the opening 92 a reaches the flow path 95 through the cover layer 94. As a result, the fluorescent sample held in the channel 95 is excited and emits fluorescence 96. This fluorescence 96 becomes divergent light, and at least a part of them reaches a photodetector (not shown) and is detected.

  Here, the incident light 98 is irradiated so that the width (diameter) of the incident light 98 (transmitted light) that has reached the flow path 95 through the opening 92 a is substantially the same as the width of the flow path 95. Then, the incident light 98 can irradiate the entire flow path 95. The prior art described above is focused on one point of the flow path, whereas the present application is different in that the flow path 95 is irradiated with the spread incident light 98. Thereby, the sample in the flow channel 95 can be efficiently excited and stably emitted, for example, the fluorescent molecule can be stably excited with respect to the sample, and a highly reliable optical analysis can be realized. If the incident light 98 is concentrated and irradiated at one point as in the conventional example, the irradiation intensity is locally increased. For example, the fluorescent dye is deteriorated or the temperature is increased. A decrease occurs. On the other hand, since the present application can irradiate the whole flow path 95 uniformly, the irradiation intensity per unit area can be lowered to prevent the deterioration of the fluorescent dye and the temperature rise.

  That is, in the analysis substrate 151, the light shielding layer 92 shields the scattered light and autofluorescence from the condensing lens 100 and allows the incident light 98 to be transmitted efficiently. Further, since the width of the incident light 98 that has passed through the opening 92a is expanded in the flow path 95, and preferably is substantially the same as the width of the flow path 95, the incident light 98 (excitation light) is transmitted to the entire flow path. The sample can emit light of, for example, fluorescence 96 stably. Thereby, the autofluorescence of the condensing lens 100 and mixing of scattered light can be suppressed, and the sample can emit light stably (for example, fluorescence) and can be detected with high sensitivity.

  Further, when the incident light 98 is condensed so that the focal point of the incident light 98 coincides with the surface of the light shielding layer 92, the opening 92a can be minimized. That is, the incident light 98 can be blocked as much as possible from the scattered light and the self-emission from the objective lens 100 while passing the light as it is.

Here, in order to irradiate the incident light 98 over the entire width direction of the flow path 95 of the analysis substrate 151, the width of the flow path 95 shown in FIG. 12 is W, the depth is D, and the flow from the opening 92a. If the distance to the road 95 is G,
0.4G ≦ W ≦ 1.6 (G + D)
Each value is preferably set so as to satisfy the following condition.

  This is when the NA (numerical aperture) of the objective lens 100 is a practical value of about 0.45 (compact disc) to 0.85 (Blu-ray disc) and the refractive index of the cover layer 94 is 1.6. It corresponds to. At this time, the spread angle of the incident light 98 in the cover layer 94 is 16 to 32 degrees. This condition will be described geometrically with reference to FIG.

  When the objective lens 100 (condenser lens) is used to focus the incident light 98 on the opening 92a, the spread angle of the light beam of the incident light 98 from the focus with respect to the direction perpendicular to the incident side surface of the substrate 91 is set. If the minimum θ1 = 16 degrees and the maximum θ2 = 32 degrees, and the outer edge of the light beam of incident light passes through the side surface of the flow path 95 within this range, the sample emits fluorescence efficiently. When this is replaced with a formula using the width W of the flow path, the depth D of the flow path, and the distance from the opening to the flow path (thickness of the cover layer) G, the following is obtained.

First, the condition that the spreading edge of the incident light (outer edge of the light beam) is lower than the uppermost part of the side surface of the flow path 95 is as follows:
(W / 2) / (D + G) ≦ tan θ1 = 0.29 (1)
In the case of Blu-ray Disc,
(W / 2) / (D + G) ≦ tan θ1 = 0.63 (2)
It is. Of these two conditions (1) and (2), the wider one of the incident light 98 is expressed by equation (2).

Next, the spread diameter (outer edge of the light beam) of the incident light 98 applied to the surface of the substrate 91 where the flow path 95 is formed (the incident surface of the incident light 98 of the flow path 95) is equal to or smaller than the width of the flow path 95. In the case of a compact disc,
(W / 2) / G ≧ tan θ2 = 0.29 (3)
In the case of Blu-ray Disc,
(W / 2) / G ≧ tan θ2 = 0.63 (4)
It is. Of the two conditions (3) and (4), the narrower width of the incident light 98 is expressed by the equation (3).

Therefore, finally, the conditions of Equations (2) and (3) are summarized:
0.58G ≦ W ≦ 1.26 (G + D) (5)
It becomes.

  In other words, the above condition is expressed by the width of the incident light 98 not more than the width of the flow path 95 on the incident side surface of the incident light 98 of the flow path 95 (the lower end position of the flow path 95 in FIG. 12). The width of the incident light 98 is set to be equal to or larger than the width of the flow path 95 on the surface on the emission side of the incident light 98 of the flow path 95 (the upper end position of the flow path 95 in FIG. This is a condition for the incident light 98 to irradiate the entire flow path 95 uniformly obtained by the inventors of the present application. If this condition is satisfied, the sample of the flow path 95 is efficiently excited by the incident light 98. it can. Further, the above condition is that the width (diameter) of the incident light 98 that irradiates the cell is equal to or greater than one of the width of the incident-side surface and the emission-side surface of the cell, and the other It can also be expressed as less than the width of.

In addition, when this inventor experimented, it is preferable to have a margin of about 30% with respect to Formula (5) in both an upper limit and a lower limit. Therefore, considering this point, equation (5) is
0.4G ≦ W ≦ 1.6 (G + D) (6)
It becomes.

  An example in which fluorescence was efficiently excited from the sample in the channel 95 was G = 50 μm, W = 100 μm, D = 30 μm, and NA of the objective lens was 0.6.

  On the other hand, the incident-side objective lens 100 with respect to the analysis substrate 151 emits scattered light and weak autofluorescence 97 when incident light 98 passes through. These unnecessary lights are divergent light, and are mostly shielded by the light shielding layer 92, so that they do not reach the light detector 115. That is, the incident light 98 can pass through the opening 92 a in the light shielding layer 92 and irradiate the flow path 95, but scattered light and autofluorescence 97 are shielded by the light shielding layer 92. That is, the light that reaches the detection side of the analysis substrate 151 is only the transmitted light 99 and the fluorescence 96 from the sample generated in the flow channel 95.

  Further, if the photodetector 115 is arranged outside the optical path of the transmitted light 99, the transmitted light 99 does not enter the photodetector 115 and does not become background light, so that the fluorescence 96 can be detected with high sensitivity. it can.

  FIG. 13 is a diagram showing a light detection device (optical pickup) 110 in the analysis device that detects fluorescence of a sample from the analysis substrate 151 in FIG. The light source 117 is a semiconductor laser, for example. The configuration of the light detection device 110 is the same as that described in FIG.

  According to the above configuration, the light detection device 110 provided in the analyzer is disposed outside the optical path of the transmitted light 99. Therefore, since each optical component (111 to 113) is not irradiated with the transmitted light 99, no self-emission or scattered light is emitted therefrom. If these optical components are irradiated with the transmitted light 99, the transmitted light 99 has a very strong light intensity, so that the optical component exposed to the transmitted light 99 emits autofluorescence or scattered light. These unnecessary lights have a spectrum spread and may be detected by passing through the optical filter 112. Further, the transmitted light 99 itself may slightly leak from the optical filter 112 and enter the photodetector 115. These leakage lights become background light, but can be suppressed by disposing the light detection device 110 outside the optical path of the transmitted light 99 as described above.

  In addition, a so-called confocal optical system that allows only light passing through the focal point of the lens 113 to pass through the pinhole 114 is formed. Therefore, only fluorescence 96 from the sample to be measured can be guided to the photodetector 115. That is, unnecessary light (such as stray light) remaining other than the fluorescence 96 does not focus on the pinhole 114 in the confocal optical system, and thus is not blocked and detected.

  However, if the light receiving surface of the photodetector 115 is very small, the pinhole 114 can be omitted because the pinhole can also be used. Further, when the light receiving area of the photodetector 115 is large, the lens 111, the lens 113, and the pinhole 114 for condensing fluorescence may be omitted. Further, when unnecessary scattered light or fluorescence passing through the opening 92a is small enough to be ignored, the optical filter 112 may be omitted.

  As described above, the analysis substrate 151 of the present invention includes the substrate 91 provided with the flow path 95 for holding the sample solution, and the cover for holding the liquid in the flow path 95 provided on the formation surface of the flow path 95. A layer 94 and a light shielding layer 92 formed on the cover layer 94 are provided.

  According to said structure, the flow path 95 which exists in the board | substrate 91 is closed by the cover layer 94, and the light shielding layer 92 provided with the opening part 92a in the surface on the opposite side is formed. In this structure, when the incident light is collected by the condensing lens 100 and enters through the opening 92a, the scattered light and the autofluorescence of the objective lens 100 are easily blocked by the light shielding layer 92. It does not reach the opposite detection side across 151.

  On the other hand, the condensed incident light 98 passes through the opening 92a. The transmitted light 99 uniformly illuminates the entire flow path 95 and excites the fluorescent sample efficiently. That is, the light shielding layer 92 shields the autofluorescence 97 of the objective lens 100, while efficiently transmitting the incident light 98 through the opening 92a.

  Accordingly, only the transmitted light 99 from the incident light 98 is irradiated to the flow path 95, and only the sample fluorescence 96 generated in the flow path 95 reaches the detection side. Accordingly, it is possible to detect only the fluorescence 96 of the sample with high sensitivity while suppressing the scattered light from the objective lens 100 and the autofluorescence 97 from being mixed.

  The analysis apparatus of the present invention is located on the opposite side of the light source 117 across the analysis substrate 151, the light source 117 for making incident light incident on the analysis substrate 151, and the transmitted light. And a photo detector 115 arranged in other than 99 optical paths. Furthermore, since the photodetector 115 is disposed outside the optical path of the transmitted light 99 from the incident light 98, the transmitted light 99 does not directly enter the photodetector 115, and is caused by the transmitted light 99. Background light can be kept low.

  Therefore, according to the present invention, it is possible to provide an analysis substrate and an analysis apparatus for detecting an optical change from a sample or a reagent with high sensitivity.

  FIG. 14 shows data when a DNA sample is measured using the analysis substrate 151 and the analysis apparatus. Since the analysis substrate 151 includes the light shielding layer 92, it can be seen that the change in the detection intensity with respect to the change in the DNA concentration increases and the sensitivity increases as compared with the case where the light shielding layer 92 is not provided. Further, the limit of sensitivity is 7 ng / μl (microliter), and practical sensitivity is obtained. It should be noted that the sensitivity increases even when a commercially available pinhole plate is used instead of the light shielding layer 92, but it is understood that the light shielding layer 92 has higher sensitivity.

  The electrophoresis method in the flow channel 95 of the analysis substrate 151 will be described in detail below with reference to FIG.

  FIG. 15 shows an example in which the configuration of the main part of the analysis substrate 151 described above is applied to the disk-shaped substrate 152 and analysis using electrophoresis is performed. The disk-like substrate 152 for analysis can perform analysis using electrophoresis. Like the disk-like substrate 30 shown in FIG. 4, the center hole 31, four analysis chips 32, and this are used. Each of the liquid reservoir / injection ports 34a to 34d, the first and second migration paths 35a and 35b, and the guide groove 5 are provided. The filled portion indicates a portion where the light shielding layer 92 is present. Therefore, the light shielding layer 3 is not formed in the region overlapping with the first migration paths 35a and 35b. That is, the slit 36 which is an opening for transmitting incident light is formed in this portion, and the cross-sectional view of the portion is as shown in FIG.

  The configuration of the analysis chip 32 for electrophoresis is the same as that of the disk-shaped substrate 30. The second migration path 35b is shorter than the first migration path 35a.

  The analysis process (electrophoresis process) on the disk-shaped substrate 152 is the same as that of the disk-shaped substrate 30. The buffer / gel solution is injected from any one of the liquid reservoir / injection ports 34a to 34d shown in FIG. 15, and filled so that bubbles do not occur in the first migration path 35a and the second migration path 35b.

  In the analysis process, the first migration path 35a is efficiently irradiated with incident light as shown in FIG. 11 which is a cross-sectional view of this part, and the sample passes through the fluorescently labeled sample, so that the sample emits fluorescence. To emit. At this time, as described above, among the scattered light and autofluorescence emitted by the objective lens 100, only a few pass through the slit 36 (opening 92a in FIG. 11), and leakage to the detection side can be suppressed.

  Further, by using the light detection device 110 shown in FIG. 13, it is possible to suppress the background noise light due to the transmitted light 99 and the autofluorescence in the substrate 90. Therefore, only the fluorescence of the sample can be detected with high sensitivity.

  A function by providing the guide groove 5 for guiding incident light and the address information recording unit 33 in which address information is recorded in the disk-shaped substrate 152, a function by shaking the four analysis chips 32, etc. Is the same as in the case of the disk-shaped substrate 30.

  FIG. 16 is a block diagram showing the configuration of the main part of the analyzer 161 for the analysis substrate (analysis disk) 152 in the present embodiment. The analysis device 161 includes an optical pickup 51, an address reproduction circuit 52, a controller 53 (tracking means), a servo circuit 54 (tracking means), and a photodetector 110, as in the analysis device 50 described above. This is the same as the analyzer 50. In FIG. 16, the photodetector 110 is also specified.

  In addition to the lens 100 and the actuator 51a, the optical pickup 51 has an optical system 51b composed of optical components such as a semiconductor laser, a photodetector, and a beam splitter. In the figure, the lens 100 and the actuator 51a are components that constitute the optical pickup 51, but are illustrated separately from the optical system 51b for the sake of clarity.

  In the incident light control device 161, the incident light emitted from the optical system 51b is collected by the lens 100 and is incident on the disk-shaped substrate 152 (analysis disk). This incident light is applied to the guide groove 5 or the address information recording unit 33 of the disk-shaped substrate 152. The reflected light from the guide groove 5 or the address information recording unit 33 returns to the optical pickup 51 again and outputs a focus error signal / track error signal 55 from the optical system 51b.

  The focus error signal / track error signal 55 is fed back to the actuator 51a via the servo circuit 54 and controls the actuator 51a so that the incident light is guided to the guide groove 5 or the address information recording unit 33. The light quantity detection signal 56 is input to the address reproduction circuit 52, detects address information 57, and sends the address information 57 to the controller 53.

  The controller 53 outputs a control signal 58 to the servo circuit 54 while confirming the address information 57, and performs a process of allowing incident light to access a guide groove at a desired address. Thereby, the incident light is moved to the radial position on the disk-shaped substrate 152, and fluorescence detection can be performed at a desired position along the flow path 35.

  Further, the disk-shaped substrate 152 is rotated, and fluorescence detection is performed with an analysis chip having a desired number. In addition, on the flow path 35 without the guide groove 5, the tracking state in the immediately preceding guide groove is maintained. In order to maintain the time, it is sufficient that the time traversing the flow path 35 is sufficiently shorter than the response time of the servo (proportional to the inverse of the servo band). Or what is necessary is just to hold | maintain servo operation temporarily, when crossing the flow path 35 (hold operation | movement).

  By doing so, the tracking is not disturbed even if the incident light 98 crosses the flow path 35, and the sample is irradiated with the incident light 98 accurately, and then reaches the guide groove 5 again to return to the tracking operation. be able to.

  The disk-shaped substrate 152 has a light shielding layer 92 that suppresses scattered light from the lens 100 and autofluorescence 97, and the detector 110 is disposed outside the optical path of the transmitted light 99. The fluorescence intensity detected by the detector 110 is converted into an electric signal 60 and sent to the controller 58.

  In addition, although the slit 36 was used as the opening part 92a, you may use not only this but micro opening parts, such as a pinhole. When the slit 36 along the flow path 35 is used for the opening 92a as in the present embodiment, fluorescence detection at an arbitrary position of the flow path 35 is possible. In addition, when a pinhole is used as the opening 92a, autofluorescence / scattered light from the lens and the cover layer reaching the detector can be reduced as compared with the case of the slit.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.

  In each of the above-described embodiments, an example in which the wavelength of incident light is long and scattered light is mainly reduced by the light shielding layer is shown. However, the present invention is not limited to this, and it goes without saying that even when the wavelength of the incident light is relatively short and the self-emission is stronger than the scattered light, the light-shielding layer can reduce the self-emission.

  In the analysis substrate and the analysis apparatus of the present invention, when optically analyzing a sample solution, the amount of background noise light caused by incident light reaching the photodetector can be reduced, so that the sensitivity of optical detection is increased. Therefore, it can be suitably used in the field of analyzing a very small amount of sample.

It is sectional drawing of the state in which incident light injected into the board | substrate for analysis as described in Example 1 of this invention. FIG. 2 is a schematic front view of a light detection device that detects fluorescence emitted from a sample in a flow path of an analysis substrate illustrated in FIG. 1. It is sectional drawing of the state in which incident light injected into the board | substrate for analysis as described in Example 2 of this invention. It is a top view which shows the disk-shaped board | substrate for analysis to which the structure of the board | substrate for analysis shown in FIG. 3 is applied. FIG. 4 is a longitudinal sectional view showing a state where incident light for analysis passes through a flow path and moves to a tracking region of a guide groove in the analysis substrate shown in FIG. 3. It is a block diagram which shows the structure of the principal part of the incident light control apparatus in this Embodiment. It is sectional drawing of the state in which incident light injected into the board | substrate for analysis as described in Example 3 of this invention. FIG. 8 is a schematic front view of a light detection device that detects fluorescence emitted from a sample in the flow path of the analysis substrate shown in FIG. 7. It is a schematic plan view which shows Example 4 of the board | substrate for analysis of this invention. It is a longitudinal cross-sectional view of the conventional analysis chip. It is sectional drawing of the state in which incident light injected into the board | substrate for analysis as described in Example 5 of this invention. It is a figure explaining the optimal structure of the flow path of the board | substrate for analysis in FIG. FIG. 12 is a schematic front view of a light detection device that detects fluorescence emitted from a sample in the flow path of the analysis substrate illustrated in FIG. 11. It is a graph which shows the experimental data at the time of using the board | substrate for analysis shown in FIG. It is a top view which shows the disk-shaped board | substrate for analysis to which the structure of the board | substrate for analysis as described in Example 6 of this invention is applied. FIG. 10 is a block diagram illustrating a configuration of a main part of an analyzer according to a sixth embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Analysis substrate 2 Substrate with flow path 3 Light shielding layer 3a Opening 4 Protective layer 5 Guide groove 6 Adhesive layer 7 Cover layer 8 Channel 9 Guide groove 10 Fluorescence from sample 11 Autofluorescence 12 Incident light 13 Transmitted light 14 Lens
DESCRIPTION OF SYMBOLS 30 Analysis disk 31 Fixing center hole 32 Analysis chip 33 Address information recording part 34a-d Liquid reservoir / injection port 35a, b Electrophoretic path 36 Slit 41 Reflected light 50 Incident light control apparatus 51 Pickup 51a Actuator 51b Other pick-up components 52 address reproduction circuit 53 controller 54 servo circuit 55 focus signal / track error signal 56 light detection signal 57 address information 58 control signal 60 analysis substrate 61 substrate with flow path 62 light shielding layer 62a opening 63 protection layer 64 guide groove 65 adhesion Layer 66 Cover layer 67 Flow path 68 Guide groove 69 Fluorescence from sample 70 Autofluorescence 71 Incident light 72 Transmitted light 73 Lens 74 Photodetector schematic diagram 75 Lens for condensing light from sample and converting it into parallel light 76 Optical filter 77 Lens that collects parallel light 78 Photodetector 79 Optical path collected by optical system 80 Analysis chip 81 Microchannel 82a Incident side pinhole 82b Detection side pinhole 83a Incident side microlens 83b Detection side microlens 84a Substrate 84b Detection side substrate 85 Incident light 86 Detection Fluorescence 87 Excitation light transmitted through sample 91 Substrate 92 Light shielding layer 94 Cover layer 95 Cell 90 Analysis substrate 91 Substrate with flow path 92 Light shielding layer 92a Opening portion 93 Adhesive layer 94 Cover layer 95 Flow path 96 Fluorescence from sample 97 Autofluorescence 98 Incident light 99 Transmitted light 100 Objective lens 110 Photodetector 111 Lens 112 that collects light from the sample and converts it into parallel light 112 Optical filter 113 Lens 114 that collects parallel light 114 Pinhole 115 Photodetector 116 Optical Light path 117 collected by the system

Claims (9)

In an analysis substrate capable of optical analysis of a sample solution,
A substrate with a cell for holding the sample solution;
A light shielding layer formed on the substrate surface provided with the cell;
An analysis substrate, comprising: a cover layer that is provided on a surface of the substrate on the cell formation side and covers the cell so that liquid is maintained in the cell.   The analysis substrate according to claim 1, wherein the light shielding layer includes an optical opening.   The analysis substrate according to claim 1, further comprising a protective layer covering the light shielding layer. In an analysis substrate capable of optical analysis of a sample solution,
A substrate with a cell for holding the sample solution;
A light shielding layer made of a reflective film formed on the substrate surface;
A cover layer that is provided on a surface of the substrate on which the cell is formed and covers the cell so that liquid is maintained in the cell;
An analysis substrate, comprising: guide means for guiding the incident light for optical analysis at the position of the cell formed on the substrate.   5. The analysis substrate according to claim 1, wherein the substrate on which the cells are engraved is a light incident side substrate, and the cover layer is a light emission side substrate.   The light shielding layer is provided so as to cover the entire surface of the substrate including the cell formation region on the side where the cells are formed, and is present on the incident side of incident light for optical analysis than the light shielding layer. The analysis substrate according to claim 1, wherein self-emission from a member to be blocked is blocked and incident light for optical analysis having a higher intensity than the self-emission is transmitted. A light source for making incident light incident on the analysis substrate according to claim 1;
An analysis apparatus comprising: a photodetector located on a side opposite to the light source across the analysis substrate and disposed at a position off the optical path of the incident light. An optical pickup having a light source for making incident light incident on the analysis substrate according to claim 4, and detecting reflected light from the analysis substrate;
Moving means for moving the optical pickup in a direction along the guide means;
An analyzer comprising: tracking means for controlling the optical pickup so that the incident light follows the guide means based on a detection signal from the optical pickup.   The analyzer according to claim 7, further comprising a pinhole between the photodetector and the analysis substrate.

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