JP2003294754A - Support body for fluorescence detection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a support body structure capable of efficiently detecting fluorescence without intensifying excitation light nor changing detection optical systems such as a photoelectron multiplier. <P>SOLUTION: Specimens labelled by means of fluorescent pigment excited by incident light of wavelength λ are immobilized. A thin film comprising at least one layer is formed on a surface of a base material for structuring a fluorescence detection support body for detecting fluorescence from the fluorescent pigment by irradiating it with the incident light, so that the optical film thickness nd (n for flexibility, d for film thickness) of the thin film falls within a range of an odd number of times of λ/4±λ/10. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a fluorescent detection support for detecting a fluorescently labeled sample.

[0002]

2. Description of the Related Art In recent years, techniques for highly sensitive detection of biological substances have been actively reported, and in particular, studies on nanoscale test samples such as genes have become possible.
For example, analysis by a nucleic acid probe array is performed as a method of detecting the expression frequency of a gene to be detected. Conventionally, a nylon membrane has been used as a support for detection by nucleic acid hybridization reaction,
The probe DNA was immobilized on the substrate by utilizing the + charge of the above (Japanese Patent Application No. 10-311164). The detection after the hybridization reaction is performed by chemiluminescence or radioisotope.

In recent years, a method of immobilizing various kinds of probe DNAs at a high density using a slide glass (about 7.5 cm × 2.5 cm) of a certain standard by the microarray technology (Japanese Patent Application No. 11-189360). Gazette) and a method of synthesizing a DNA probe on a semiconductor substrate by a photolithography technique. By using these microarray techniques, one spot was 3 mm to 5 mm in the nylon film, but 100 spots in the glass substrate.
Ultra-fine spots of μm to 300 μm are possible, and high density can be achieved. Further, unlike the nylon film, since the glass or silicon substrate has little fluorescence, it is possible to detect the hybridization result by fluorescence detection.

[0004]

As described above, in recent years,
In the analysis of DNA, a large number of different DNA probes are put on a solid substrate such as glass in a range of 100 μm to 300 μm.
Is immobilized at a high density with a very small spot size, labeled DNA is hybridized on it, the signal from each probe is detected by an automatic detector, and the data is analyzed in large quantities by a computer. However, this has the following problems.

Compared with a nylon film, a glass or silicon substrate has a smaller surface area capable of reacting, so that the amount of immobilized probe DNA is smaller. On the other hand, in measuring the expression frequency of a gene, a gene with a low expression frequency can obtain a very weak fluorescence intensity. Therefore, higher-sensitivity detection is desirable for high-accuracy measurement, but current technology that combines a confocal optical system and a photomultiplier tube may not be enough to achieve near-limit high sensitivity. is there. Further, a glass substrate which has been commercially available as a fluorescence detection substrate for a microarray in the related art is relatively expensive because it is subjected to a special surface polishing treatment or the like to improve its sensitivity.

Therefore, the present invention intends to provide a support structure which enables efficient fluorescence detection without changing the detection light system such as enhancement of excitation light or photomultiplier tube and which can be manufactured at low cost. To do.

[0007]

The support for fluorescence detection of the present invention is such that a sample labeled with a fluorescent dye that is excited by incident light of wavelength λ is fixed, and the fluorescence from the fluorescent dye is irradiated by irradiation of the incident light. Which is a support for detecting fluorescence, in which a thin film composed of at least one layer is formed on the surface of a base material, and an optical film thickness nd (n is a refractive index, d is a film thickness) of the thin film. ) Is an odd multiple of λ / 4 ± λ / 10.

The transmittance of incident light of the thin film is preferably 90% or more, and specifically, the thin film is preferably a single-layer or multi-layer insulating coating.

The base material preferably has a refractive index higher than that of the thin film and a reflectance to incident light of 80% or more. Specifically, the base material is selected from the group consisting of silicon, aluminum, chromium and gold. It is preferably formed of at least one metal or semiconductor selected. Further, the surface of the base material may have a groove structure.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors, when a fluorescently labeled sample is immobilized on a support having a multilayer film structure and fluorescence is detected,
It was found that the fluorescence intensity is enhanced at a specific film thickness. Although the reason for this is not clear, it was revealed from the relationship between the incident light and the film thickness that a high fluorescence intensity is exhibited under the condition where the reflected light intensity is the lowest. The present invention is based on this finding.

Hereinafter, embodiments of the present invention will be described with reference to the drawings, but these are not intended to limit the present invention, and various substitutions, additions, and other changes can be made in accordance with the gist of the present invention. .

FIG. 1 shows a schematic diagram (FIG. 1a) showing an embodiment of a support of the present invention, and a principle diagram (FIG. 1b) when fluorescence excitation is performed using the support. Figure 1a
Shows that a thin film 2 is formed on a substrate 1 and a sample 3 labeled with a fluorescent dye is fixed on the thin film 2.

Here, as described above, it has been known that a high fluorescence intensity can be obtained under the condition of the lowest reflection intensity, but the principle is considered as follows. Figure 1b
Shows that the support of FIG. 1a was irradiated with excitation light to excite fluorescence. When the excitation light of fluorescence (incident light 4) is incident on the support, under the condition that the product of the film thickness d and the refractive index n is an odd multiple of λ / 4 with respect to the wavelength λ of the incident light 4, It is assumed that multiple reflections 5 occur between the surface and the surface of the thin film 2. At this time, the incident lights 4 are confined in the thin film 2 and are mutually strengthened by the interference action, and the sample 3 is irradiated with the incident lights 4. Therefore, it is assumed that the incident light 4 reflected by the support is efficiently used and the higher intensity fluorescence 6 is obtained. That is, even if the same detection optical system is used, high sensitivity can be expected.

The size of the support of the present invention is not particularly limited. For example, in the case of a strip-shaped support, a slide glass of a certain standard (about 7.5 cm × 2.
It is preferably 5 cm).

The thin film constituting the support of the present invention preferably uses an insulating coating in order to prevent quenching of fluorescence emitted from the fluorescent substance. The transmittance of incident light of the thin film is preferably 80% 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 has high transparency and does not emit fluorescence.
It is not limited to these. The thin film is formed by a method used for forming an ordinary thin film, for example, thermal oxidation. The film thickness is adjusted so that the intensity of reflected light becomes the minimum condition. That is, the optical film thickness nd of the thin film (n is the refractive index of the material, d is the thickness of the thin film) is λ /
It is adjusted so that the range is an odd multiple of 4 ± λ / 10. The film thickness is preferably 1 mm or less.

The thin film is formed on at least a part of the surface of the base material, and may be formed on the entire surface. For example, if the support of the present invention is to be used in a DNA array, DN
The A probe may be locally formed in the portion where the solid phase is fixed. Strictly speaking, it is preferable to consider the fixed object fixed on the surface by including it in the optical film thickness, but it is difficult to control the film thickness in reality, 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, the 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, and for example, a nucleic acid containing a gene, an antigen,
Antibodies, proteins containing enzymes, allergens, physiologically active substances containing hormones, cells, etc. can be used. Even if the object to be immobilized is other than nucleic acid such as DNA, the object to be immobilized can be immobilized with any thickness and density as long as it has a certain degree of light transmittance.

Further, as an application example of the present invention, the optical film thickness nd (product of refractive index and film thickness) is λ with respect to the wavelength λ of incident light.
By using one or more supports provided with a plurality of different film thickness portions in the range of an odd multiple of / 4 ± λ / 10, an object to be fixed different for each film thickness can be fixed or each film thickness can be fixed. By reacting with different reaction reagents and / or different test samples, it is possible to test multiple items with high throughput.

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. Further, even those having light absorption characteristics can be essentially applied to the present invention. In some cases, it may have a light-transmitting property so that the transmitted light can be used, but the light-reflecting one causes the multiple reflections in the thin film on the substrate to increase the fluorescence intensity as described above. It is preferable because it can be enhanced. That is, the reflectance of incident light on the surface of the base material is preferably 80% or more, and it is more preferable to use one having a high reflectance. As a specific example, a surface-polished semiconductor such as silicon, or a surface-polished metal base material such as aluminum, chromium, or gold can be used, but is not limited thereto.

The thin film and the base material of the support of the present invention are not limited to one layer and have the lowest reflectance, that is, the optical film thickness nd of the thin film is λ with respect to the wavelength λ of the incident light. /
A multilayer structure may be used as long as it satisfies the condition of being an odd multiple of 4 ± λ / 10.

The shape of the support of the present invention is not particularly limited as long as it has a refractive index larger than that of the thin film, as long as it can be used for immobilizing an object to be immobilized on the surface and detecting fluorescence. The surface of the support does not have to be flat, and may have a groove shape as shown in FIG. 2 or a convex structure. The groove structure as shown in FIG. 2 can be used as a flow channel structure by covering the upper part thereof with a cover. Further, the surface of the support may have a curved surface, and a flow path having an inner wall having a circular or elliptical cross section may be formed. Furthermore, the support may be in the form of particles such as spheres or polyhedra. In this case, various detections in which one or more particulate supports are suspended or settled in a liquid and the motion state of the particles is taken into consideration (for example,
Particle agglutination analysis, fluorescence correlation spectroscopy, etc.) are also possible, and a homogeneous mixture can be obtained. 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 end face thereof.

With the above structure, the incident light can be effectively used and the fluorescence can be detected with high sensitivity.
The support of the present invention can be effectively used for detecting a trace amount of sample such as a DNA probe array. further,
By selecting the materials used for the base material and the thin film, a support for fluorescence detection having a higher sensitivity than the conventionally used slide glass for microarray can be manufactured at a much lower cost.

[0022]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto.

<Embodiment 1> FIG. 3 shows that silicon oxide films of various thicknesses are formed on the surface of a silicon substrate (refractive index: n = 4.09, k = 0.04). The reflectance obtained by irradiating the incident light at an incident angle of 90 degrees is calculated. The vertical axis represents the reflectance and the horizontal axis represents the film thickness of the silicon oxide film. Here, the wavelength of the incident light is 543
The reason for using nm is that the wavelength of the excitation light of the device used for the fluorescence measurement in the following examples was used. Although this result is a calculation result, the actual reflectance measurement result also showed the same result. From this result, the refractive index of the silicon oxide film n: 1.4
The product nd of 6 and the film thickness d is approximately λ / 4 of the wavelength λ of the incident light.
It was found that the reflectance was the lowest under the condition that the refractive index of the substrate was larger than that of the thin film at an odd multiple of. This is a condition similar to the condition nd = λ / 4 of 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.

Example 2 A surface-polished silicon substrate having a flat surface (diameter: 4 inches, thickness: 0.5 mm, (10
Silicon oxide films of 100 nm and 200 n, respectively, are formed on single crystal silicon (0) plane (Semitech Co., Ltd.).
m, 300 nm, 400 nm, 480 nm thickness formed substrate of DNA probe solid phase was prepared. The oxide film is
After the surface of the silicon substrate was RCA cleaned, it was formed in an oxidation furnace so as to have a predetermined film thickness. Here, the RCA cleaning is
80 with a mixture of hydrogen peroxide: hydrochloric acid: pure water = 1: 1: 6
After boiling at 10 ° C. for 10 minutes, the mixture was boiled at 80 ° C. for 10 minutes with a mixed solution of hydrogen peroxide: ammonia water: pure water = 1: 1: 6, and washed with running pure water. For comparison, a commercially available slide glass (white glass) and a microarray slide glass (MA) were prepared. A DNA probe was solid-phased on these substrates by the procedure shown below, and after the hybridization reaction, fluorescence observation was performed.

As a slide pretreatment method, a glass substrate is generally washed with an alkali or an acid. afterwards,
Perform necessary processing on the probe DNA solid phase. In this experiment,
In order to prevent the thickness of the silicon oxide film from changing, cleaning was performed with a mixed solution of weak alkali and hydrogen peroxide. The surface was silanized using a 1% dilute solution of γ-aminopropyltriethoxysilane to form amino groups. After rinsing this with pure water and drying it, SPB which is a pin type arrayer
The probe DNA was spotted using IO (manufactured by Hitachi Software Engineering Co., Ltd.). A linker molecule having sites for binding amino groups on both sides is mixed with the probe solution, and the amino group on the surface of the substrate and the amino group at the terminal of the DNA probe are covalently fixed by this linker molecule. The probe DNA immobilized on the substrate side was 5'NH-cat-GAC TGA ATT GTT TTT.
TGC TCA TAC CCT 3 '(30mer)
It is an oligonucleotide probe having the sequence of. After the slide was prepared, a sequence complementary to the probe DNA (5 ′ AGG GTA TGA GCA AAA A
A hybridization reaction was carried out by using an oligonucleotide having AC AAT TCA TGC ATG 3 ') labeled with a 5'-end fluorescence as a target DNA. The fluorescent dye used this time is Cy3, and the wavelength is 535.
It is a dye that emits fluorescence of 560 nm with excitation light of nm. 1x slide after hybridization reaction
After washing with SSC (standard salt-citrate buffer), a fluorescence detector ScanArray4000 (GSI lumon) was used.
ics). However, the fluorescence detector Sc
The anArray4000 has a wavelength of 543 nm due to the device configuration.
Of excitation light was used. Two experiments were conducted for each substrate having each film thickness, and the fluorescence intensity of each substrate was obtained. The fluorescence intensity is 64
It is the average value of the spots. The measured values of the observation results are shown in Table 1. In addition, FIG. 4 is a graph showing the results of Table 1 with the fluorescence intensity taken on the vertical axis.

[0026]

[Table 1]

A silicon oxide film is formed from 100 nm to 480 n
When it was changed to m, a periodic intensity change was shown as shown in the figure. Further, the maximum value of the fluorescence intensity was several times that of commercially available white glass and slide glass for microarray. Comparing this result with FIG. 3, it was found that almost the same periodic change was observed, that is, the higher the excitation light reflectance, the higher the fluorescence intensity obtained. This is considered to be because the fluorescent dye can be efficiently excited by the principle shown in FIG. 1b when the excitation light is used under the condition of low reflectance.

<Embodiment 3> Next, in order to carry out a more detailed study on the detection result of Embodiment 2, the thickness of the silicon oxide film is finely changed to 60 nm, 80 nm and 100 nm.
The experiment was performed using a substrate for a DNA probe solid phase having a wavelength of 120 nm, 120 nm, or 140 nm. The other experimental conditions are the same as those described in Example 2. The result is shown in FIG. The fluorescence intensity reached a maximum at a film thickness of 80 nm. In the reflectance measurement of FIG. 3, the minimum value was shown at about 90 nm, and although there was some deviation, it showed the same tendency as in FIG. 4, so that the reflectance and the fluorescence intensity change in the same cycle. Conceivable.

From these experimental results, it has been found that higher sensitivity fluorescence observation is possible by forming the support for fluorescence observation into a multi-layer structure to form a thin film that minimizes the reflectance of excitation light. It was

Calculating from the results of Examples 1 to 3 above, in the silicon oxide film and the silicon substrate structure, if the reflectance is about 30% or less, the fluorescence intensity is higher than that of the commercially available white glass or slide glass for microarray. Was observed, and at higher reflectances, on the contrary, worse results were obtained than those on the market. If this result is applied to a general structure, by setting the optical film thickness nd to be an odd multiple of λ / 4 within ± λ / 10, good results can be obtained as compared with commercially available white glass and slide glass for microarray. I understood.

[0031]

As described in detail above, according to the present invention,
Provided is a support structure which enables efficient fluorescence detection without increasing excitation light or changing a detection optical system such as a photomultiplier tube and which can be manufactured at low cost.

[Brief description of 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 structural diagram of a support in the case where the support for fluorescence detection of the present invention has a groove structure on the surface.

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 the fluorescence intensity when a fluorescent labeling substance on a support having silicon oxide films of various thicknesses formed on a silicon substrate is irradiated with excitation light.

FIG. 5 is a graph showing fluorescence intensity when a fluorescent labeling substance on a support having silicon oxide films of various thicknesses formed on a silicon substrate is irradiated with excitation light.

[Explanation of symbols]

1 ... Base material 2 ... Thin film 3 ... Sample 4 ... Excitation light 5 ... Multiple reflection 6 ... Synchrotron radiation

Continued front page    (72) Inventor Yoko Ohashi             2-43 Hatagaya, Shibuya-ku, Tokyo Ori             Inside Npus Optical Industry Co., Ltd. F-term (reference) 2G043 AA01 BA16 CA03 DA02 EA01                       FA01 GA07 GB01 KA05 LA01                       MA01

Claims (6)

[Claims] 1. A support for fluorescence detection 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 base material, and the optical film thickness nd (n is a refractive index, d is a film thickness) of the thin film is an odd multiple of λ / 4 ± λ / 10. A support for fluorescence detection, characterized by having a range. 2. The support for fluorescence detection according to claim 1, wherein the thin film has a transmittance of incident light of 90% or more. 3. The support for fluorescence detection according to claim 1, wherein the thin film is a single-layer or multi-layer insulating coating. 4. The fluorescence detection device according to claim 1, wherein the base material has a refractive index higher than that of the thin film, and the reflectance with respect to incident light is 80% or more. Support. 5. The substrate according to claim 1, wherein the base material is formed of silicon or at least one metal or semiconductor selected from the group consisting of aluminum, chromium and gold. The support for fluorescence detection according to item 1. 6. The support for fluorescence detection according to claim 1, wherein the surface of the base material has a groove structure.