JP4121762B2 - Fluorescence detection support - Google Patents

Description

【００２７】
シリコン酸化膜を１００ｎｍから４８０ｎｍまで変化させると、図に示すように周期的な強度変化を示した。また、蛍光強度の最大値は市販の白ガラスやマイクロアレイ用スライドガラスに比べ数倍の強度であった。この結果と図３を比較するとほぼ同様の周期的変化が認められ、すなわち励起光の反射率が低くなるほど高い蛍光強度が得られることが明らかになった。これは、反射率が低い条件で励起光を用いると、図１ｂに示した原理により効率的に蛍光色素を励起できるためと思われる。
【００２８】
＜実施例３＞
次に、実施例２による検出結果に関して更に詳細な検討を行うため、シリコン酸化膜の厚さを細かく変化させ、６０ｎｍ、８０ｎｍ、１００ｎｍ、１２０ｎｍ、１４０ｎｍとしたＤＮＡプローブ固相用基板で実験を行った。他の実験条件は実施例２に記載の条件と同一である。その結果を図５に示す。蛍光強度は８０ｎｍの膜厚で最大となった。図３の反射率の測定においては約９０ｎｍに極小値を示しており、多少のずれはあるものの、図４と同様の傾向を示したことから、反射率と蛍光強度は同様の周期で変化すると考えられる。
【００２９】
これらの実験結果から蛍光観察用支持体を多層膜構造にして励起光の反射率を最小にするような薄膜を形成することにより、より高感度の蛍光観察が可能であることが分かった。
【００３０】
上記実施例１ないし３の結果から計算すると、シリコン酸化膜とシリコン基板構造においては、反射率が約３０％以下であれば市販の白ガラスやマイクロアレイ用スライドガラスに比べて高い蛍光強度が観察され、それ以上の反射率では逆に市販のものよりも悪い結果が得られた。この結果を一般的な構造に適用すると光学的膜厚ｎｄがλ／４の奇数倍±λ／１０以内とすることにより、市販の白ガラスやマイクロアレイ用スライドガラスに比べ良好な結果が得られることが分かった。
【００３１】
【発明の効果】
以上詳述したように、本発明によれば、励起光の増強や光電子倍増管などの検出光学系を変えることなく、効率的な蛍光検出が可能であり、かつ低価格で製造できる支持体構造が提供される。
【図面の簡単な説明】
【図１】本発明の支持体の一実施の形態を示した模式図（ａ）、およびその支持体を用いて蛍光励起を行った場合の原理図（ｂ）。
【図２】本発明の蛍光検出用支持体の表面に溝構造を有する場合の支持体の構成図。
【図３】シリコン基板上にシリコン酸化物膜を形成したときの単層膜の反射率を表すグラフ。
【図４】シリコン基板上に種々の膜厚のシリコン酸化物膜を形成した支持体上の蛍光標識物質に励起光を照射した場合の蛍光強度を表すグラフ。
【図５】シリコン基板上に種々の膜厚のシリコン酸化物膜を形成した支持体上の蛍光標識物質に励起光を照射した場合の蛍光強度を表すグラフ。
【符号の説明】
１…基材
２…薄膜
３…試料
４…励起光
５…多重反射
６…放射光[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a fluorescence detection support for detecting a fluorescently labeled sample.
[0002]
[Prior art]
In recent years, highly sensitive detection techniques for biological materials have been actively reported, and research on nanoscale test samples such as genes has become possible. For example, analysis using a nucleic acid probe array is performed as a technique for detecting the expression frequency of a gene to be detected. Conventionally, a nylon membrane was used as a support for detection by a nucleic acid hybridization reaction, and probe DNA was immobilized on a substrate by utilizing the + charge of DNA (Japanese Patent Application No. 10-311164). Detection after the hybridization reaction is performed by chemiluminescence or radioisotope.
[0003]
In recent years, a method of immobilizing many kinds of probe DNAs at high density using a slide glass (about 7.5 cm × 2.5 cm) of a certain standard by microarray technology (Japanese Patent Application No. 11-189360) and A method of synthesizing a DNA probe on a semiconductor substrate by a photolithography technique is known. By using these microarray technologies, one spot is 3 mm to 5 mm in the nylon film, but a very small spot of 100 μm to 300 μm is possible on the glass substrate, and high density can be achieved. Unlike nylon membranes, glass and silicon substrates have little fluorescence, so hybridization results can be obtained by fluorescence detection.
[0004]
[Problems to be solved by the invention]
As described above, in recent years, in DNA analysis, a large number of different DNA probes are immobilized on a solid phase substrate such as glass with a very small spot size of 100 μm to 300 μm, and labeled DNA is hybridized thereon. The signal from each probe is detected with an automatic detector, and the data is mass analyzed with a computer. However, this has the following problems.
[0005]
In a glass or silicon substrate, the amount of probe DNA immobilized is small because the surface area capable of reaction is small compared to a nylon membrane. On the other hand, in the measurement of gene expression frequency or the like, a gene with low expression frequency can obtain only a very weak fluorescence intensity. For this reason, detection with higher sensitivity is desirable in order to perform high-accuracy measurement, but the current technology that combines a confocal optical system and a photomultiplier tube may not be sufficient to achieve high sensitivity close to the limit. is there. In addition, a glass substrate that is commercially available as a fluorescence detection substrate for microarrays in the past is relatively expensive because it has been subjected to a special surface polishing treatment or the like in order to improve its sensitivity.
[0006]
Therefore, the present invention intends to provide a support structure that enables efficient fluorescence detection and can be manufactured at low cost without changing the detection optical system such as enhancement of excitation light and photomultiplier tube. is there.
[0007]
[Means for Solving the Problems]
The fluorescent detection support of the present invention is a fluorescent detection support for fixing a sample labeled with a fluorescent dye excited by incident light having a wavelength λ and detecting fluorescence from the fluorescent dye by irradiation with the incident light. A thin film comprising at least one layer is formed on the surface of the substrate, and the optical film thickness nd of the thin film (where n is a refractive index and d is a film thickness) is an odd multiple of λ / 4 ± In the range of λ / 10, the base material has a refractive index larger than that of the thin film .
[0008]
Specifically, the thin film is preferably a single-layer or multilayer insulating film.
[0009]
The refractive index of the substrate is preferably larger than that of the thin film and the reflectance with respect to incident light is preferably 80% or more. Specifically, the substrate is at least selected from the group consisting of silicon, aluminum, chromium, and gold. It is preferably formed of one kind of metal or semiconductor. Further, the surface of the base material may have a groove structure.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have found that when a fluorescently labeled sample is fixed on a support having a multilayer structure and fluorescence detection is performed, the fluorescence intensity is enhanced at a specific film thickness. The reason for this is not clear, but when the relationship between the incident light and the film thickness is observed, it has been clarified that the reflected light intensity exhibits a high fluorescence intensity under the lowest conditions. The present invention is based on this finding.
[0011]
Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, these do not limit the present invention, and various substitutions, additions, and the like can be made in accordance with the gist of the present invention.
[0012]
FIG. 1 shows a schematic diagram (FIG. 1a) showing an embodiment of the support of the present invention and a principle diagram (FIG. 1b) when fluorescence excitation is performed using the support. FIG. 1a shows a state in which a thin film 2 is formed on a substrate 1, and a sample 3 labeled with a fluorescent dye is fixed thereon.
[0013]
Here, as described above, it is known that a high fluorescence intensity can be obtained under the condition of the lowest reflection intensity, but the principle is considered as follows. FIG. 1b shows a state where excitation light is applied to the support of FIG. 1a to excite fluorescence. When fluorescent excitation light (incident light 4) is incident on the support, the product of the film thickness d and the refractive index n with respect to the wavelength λ of the incident light 4 corresponds to an odd multiple of λ / 4. It is assumed that multiple reflection 5 occurs between the surface and the surface of the thin film 2. At this time, the incident light 4 is confined in the thin film 2, strengthens each other by the interference action, and is irradiated to the sample 3. Therefore, it is assumed that the incident light 4 reflected on the support is efficiently used, and higher-intensity fluorescence 6 is obtained. That is, even if the same detection optical system is used, high sensitivity can be expected.
[0014]
The size of the support of the present invention is not particularly limited. For example, in the case of a strip-shaped support, the size of a standard slide glass (about 7.5 cm × 2.5 cm) may be used due to its versatility. preferable.
[0015]
The thin film constituting the support of the present invention preferably uses an insulating coating in order to prevent quenching of the fluorescence emitted from the fluorescent material. The incident light transmittance of the thin film is preferably 80% or more, and particularly preferably 90% or more. As a specific example, a silicon oxide film, aluminum oxide or the like can be used, and a plastic thin film can be used as long as it is highly transparent and does not emit fluorescence, but is not limited thereto. . A thin film is formed by the method used for formation of a normal thin film, for example, is formed by thermal oxidation. The film thickness is adjusted so that the reflected light intensity is the minimum condition. That is, the optical film thickness nd of the thin film (where n is the refractive index of the material and d is the thickness of the thin film) is adjusted to be in the range of odd multiples of λ / 4 ± λ / 10 with respect to the incident light λ. To do. The film thickness is preferably 1 mm or less.
[0016]
The thin film may be formed on at least a part of the surface of the substrate and formed on the entire surface. For example, when the support of the present invention is to be used for a DNA array, it may be locally formed on the portion where the DNA probe is solid-phased. Strictly speaking, it is preferable to consider the fixed object fixed on the surface in the optical film thickness, but in reality, it is difficult to control the film thickness, and the thickness of the fixed object (DNA probe) is Since it is about several tens of nm at most, it can be regarded as an allowable range of the film thickness range. Here, a DNA probe is shown as an example of the immobilization target, but the immobilization target that can be used for the support of the present invention is not particularly limited as long as it can be fluorescently labeled. For example, a nucleic acid containing a gene, an antigen, Antibodies, proteins including enzymes, allergens, physiologically active substances including hormones, cells, and the like can be used. Even if the object to be fixed is other than a nucleic acid such as DNA, the object to be fixed can be fixed with an arbitrary thickness and density as long as it has a certain light transmittance.
[0017]
Further, as an application example of the present invention, a plurality of different optical film thicknesses nd (product of refractive index and film thickness) are in the range of an odd multiple of λ / 4 ± λ / 10 with respect to the wavelength λ of incident light. By using one or more supports provided with a film thickness portion, multiple fixed items can be fixed for each film thickness, or reacted with a reagent for reaction and / or a test sample different for each film thickness. There is a possibility that this inspection can be performed with a high processing capacity.
[0018]
As the base material constituting the support of the present invention, any material can be used as long as it reflects incident light on the surface on which the thin film is formed. In addition, even those having light absorption characteristics are essentially applicable to the present invention. In some cases, it may be light transmissive so that transmitted light can be used. However, as described above, multiple reflection occurs in the thin film on the base material to increase the fluorescence intensity. This is preferable because it can be enhanced. That is, the reflectance of incident light on the substrate surface is preferably 80% or more, and it is more preferable to use a material having a high reflectance. As a specific example, a surface-polishing semiconductor such as silicon or a surface-polishing metal substrate such as aluminum, chromium, or gold can be used, but is not limited thereto.
[0019]
The thin film and substrate of the support of the present invention are not limited to one layer, and the reflectance is lowest, that is, the optical film thickness nd of the thin film is λ / 4 with respect to the wavelength λ of incident light. A multilayer structure may be used as long as the condition of being an odd multiple of ± λ / 10 is satisfied.
[0020]
The shape of the support of the present invention is not particularly limited as long as the refractive index is larger than that of the thin film, as long as it is a shape that can be used for fluorescence detection by fixing an object to be fixed on the surface. The surface of the support need not be flat. For example, a groove shape as shown in FIG. 2 or a convex structure may be used. The groove structure as shown in FIG. 2 can be used as a channel structure by covering the upper part with a cover. Further, the surface of the support may have a curved surface, and may form a flow path having an inner wall whose cross section is circular or elliptical. Further, the support may be in the form of particles such as a sphere or a polyhedron. In this case, various types of detection (for example, particle agglutination analysis, fluorescence correlation spectroscopy, etc.) taking into account the motion state of particles by suspending or sedimenting one or more particulate supports in a liquid. In addition, a homogeneous mixed solution can be obtained. Further, when the support is rod-shaped, thread-shaped, fiber-shaped, tape-shaped, or strip-shaped, the object to be fixed can be fixed to the outer wall or the end surface.
[0021]
With the configuration as described above, incident light is effectively used and fluorescence can be detected with high sensitivity. Therefore, the support of the present invention is effectively used for detecting a small amount of sample such as a DNA probe array. be able to. Furthermore, by selecting the material used for the substrate and the thin film, a fluorescence detection support that is more sensitive than conventionally used glass slides for microarrays can be produced at a much lower cost.
[0022]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these.
[0023]
<Example 1>
In FIG. 3, silicon oxide films having various thicknesses are formed on the surface of a silicon substrate (refractive index: n = 4.09, k = 0.04), and incident light having a wavelength of 543 nm is incident on the surface at an incident angle of 90 degrees. The reflectance when irradiated is obtained by calculation. The vertical axis represents the reflectance, and the horizontal axis represents the thickness of the silicon oxide film. Here, the reason why the wavelength of the incident light is set to 543 nm is that the wavelength of the excitation light of the apparatus used for fluorescence measurement in the following examples is used. Although this result is a calculation result, the actual reflectance measurement result also showed the same result. From this result, when the product nd of the refractive index n: 1.46 of the silicon oxide film and the film thickness d is approximately an odd multiple of λ / 4 of the wavelength λ of the incident light, the refractive index of the substrate is larger than that of the thin film. It was found that the reflectance was the lowest. This is a condition similar to the condition nd = λ / 4 for the antireflection coating. From this, it can be seen that the silicon oxide film formed on the silicon substrate functions like a selective absorption film for incident light.
[0024]
<Example 2>
A silicon oxide film of 100 nm, 200 nm, 300 nm, and 100 nm on a flat-surface-polished silicon substrate (diameter 4 inches, thickness 0.5 mm, (100) plane single crystal silicon (Semitech Co., Ltd.)), respectively. A DNA probe solid phase substrate formed with a thickness of 400 nm and 480 nm was prepared. The oxide film was formed to have a predetermined film thickness in an oxidation furnace after RCA cleaning of the surface of the silicon substrate. Here, the RCA cleaning is performed by boiling at 80 ° C. for 10 minutes with a mixed solution of hydrogen peroxide: hydrochloric acid: pure water = 1: 1: 6, followed by mixing of hydrogen peroxide: ammonia water: pure water = 1: 1: 6. The solution was boiled at 80 ° C. for 10 minutes and then washed with pure water. For comparison, a commercially available slide glass (white glass) and a slide glass for microarray (MA) were prepared. A DNA probe was solid-phased on these substrates by the following procedure, and fluorescence observation was performed after the hybridization reaction.
[0025]
As a slide pretreatment method, a glass substrate is generally washed with alkali or acid. Thereafter, necessary processing is performed on the probe DNA solid phase. In this experiment, cleaning was performed with a mixed solution of weak alkali and hydrogen peroxide to prevent the thickness of the silicon oxide film from changing. The surface was subjected to silane treatment using a 1% diluted solution of γ-aminopropyltriethoxysilane to form amino groups. This was rinsed with pure water, and after drying, probe DNA was spotted using SPBIO (manufactured by Hitachi Software Engineering Co., Ltd.) which is a pin type arrayer. The probe solution is mixed with a linker molecule having a site that binds to an amino group on both sides, and the amino group on the substrate surface and the amino group at the end of the DNA probe are covalently fixed by this linker molecule. The probe DNA to be immobilized on the substrate side is an oligonucleotide probe having the sequence of 5′-NH-CAT GCA TGA ATT GTT TTT TGC TCA TAC CCT 3 ′ (30 mer). After the slide was prepared, a hybridization reaction was carried out using a 5'-terminal fluorescently labeled oligonucleotide having a sequence complementary to the probe DNA (5 'AGG GTA TGA GCA AAA AAC AAT TCA TGC ATG 3') as the target DNA. went. The fluorescent dye used this time is Cy3, and is a dye that emits fluorescence at 560 nm with excitation light having a wavelength of 535 nm. The slide after the hybridization reaction was washed with 1 × SSC (standard saline-citrate buffer) and detected using a fluorescence detector ScanArray 4000 (manufactured by GSI luminics). However, the fluorescence detector ScanArray 4000 used excitation light having a wavelength of 543 nm due to the device configuration. Experiments were performed on two substrates of each film thickness to determine the fluorescence intensity of each substrate. The fluorescence intensity is an average value of 64 spots. The measured values of the observation results are shown in Table 1. FIG. 4 shows the results of Table 1 in a graph with the fluorescence intensity on the vertical axis.
[0026]
[Table 1]

[0027]
When the silicon oxide film was changed from 100 nm to 480 nm, a periodic intensity change was shown as shown in the figure. Moreover, the maximum value of the fluorescence intensity was several times that of commercially available white glass and microarray slide glass. Comparing this result with FIG. 3, it was found that almost the same periodic change was observed, that is, a higher fluorescence intensity was obtained as the reflectance of the excitation light decreased. This seems to be because when the excitation light is used under a low reflectance condition, the fluorescent dye can be efficiently excited by the principle shown in FIG. 1b.
[0028]
<Example 3>
Next, in order to conduct a more detailed study on the detection results of Example 2, experiments were conducted with DNA probe solid phase substrates having a thickness of 60 nm, 80 nm, 100 nm, 120 nm, and 140 nm with fine changes in the thickness of the silicon oxide film. It was. Other experimental conditions are the same as those described in Example 2. The result is shown in FIG. The fluorescence intensity reached its maximum at a film thickness of 80 nm. In the measurement of the reflectance in FIG. 3, a minimum value is shown at about 90 nm, and although there is a slight deviation, the same tendency as in FIG. 4 is shown. Therefore, the reflectance and the fluorescence intensity change with the same period. Conceivable.
[0029]
From these experimental results, it was found that fluorescence observation with higher sensitivity is possible by forming a fluorescent observation support with a multilayer film structure and forming a thin film that minimizes the reflectance of excitation light.
[0030]
When calculated from the results of Examples 1 to 3, in the silicon oxide film and the silicon substrate structure, if the reflectivity is about 30% or less, a higher fluorescence intensity is observed as compared with commercially available white glass or microarray slide glass. On the contrary, the reflectance higher than that of the commercially available one was obtained. When this result is applied to a general structure, the optical film thickness nd is an odd multiple of λ / 4 ± λ / 10 or less, so that a better result can be obtained than commercially available white glass or microarray slide glass. I understood.
[0031]
【The invention's effect】
As described in detail above, according to the present invention, a support structure that can efficiently detect fluorescence without changing the detection optical system such as enhancement of excitation light or photomultiplier tube, and can be manufactured at low cost. Is provided.
[Brief description of the drawings]
FIG. 1 is a schematic diagram (a) showing an embodiment of a support of the present invention, and a principle diagram (b) when fluorescence excitation is performed using the support.
FIG. 2 is a configuration diagram of the support when the surface of the support for fluorescence detection according to the present invention has a groove structure.
FIG. 3 is a graph showing the reflectance of a single layer film when a silicon oxide film is formed on a silicon substrate.
FIG. 4 is a graph showing fluorescence intensity when excitation light is irradiated to a fluorescent labeling material on a support in which silicon oxide films having various thicknesses are formed on a silicon substrate.
FIG. 5 is a graph showing fluorescence intensity when excitation light is irradiated to a fluorescent labeling substance on a support in which silicon oxide films having various thicknesses are formed on a silicon substrate.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Base material 2 ... Thin film 3 ... Sample 4 ... Excitation light 5 ... Multiple reflection 6 ... Radiation light

Claims (6)

A fluorescent detection support for fixing a sample labeled with a fluorescent dye excited by incident light of wavelength λ and detecting fluorescence from the fluorescent dye by irradiation of the incident light,
A thin film consisting of at least one layer is formed on the surface of the substrate, and the optical film thickness nd (n is a refractive index and d is a film thickness) of the thin film is an odd multiple of λ / 4 ± λ / 10. A support for fluorescence detection , wherein the substrate has a refractive index greater than that of the thin film .   2. The support for fluorescence detection according to claim 1, wherein the thin film has a transmittance of incident light of 90% or more.   The support for fluorescence detection according to claim 1 or 2, wherein the thin film is a single-layer or multi-layer insulating film. The support for fluorescence detection according to any one of claims 1 to 3, wherein the base material has a reflectance of 80% or more with respect to incident light .   The said base material is formed with the at least 1 sort (s) of metal or semiconductor chosen from the group which consists of a silicon | silicone or aluminum, chromium, and gold | metal | money, The any one of Claim 1 thru | or 4 characterized by the above-mentioned. Support for fluorescence detection.   The support for fluorescence detection according to any one of claims 1 to 5, wherein the surface of the substrate has a groove structure.

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