JP2002286627A - Fluorescence detecting container - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique capable of suppressing restrictions on the design of the structure of a container and a detecting system to minimum, freely design the container, and freely using a desired adhesive regardless of the presence or absence of fluorescence when the adhesive is used. SOLUTION: This fluorescence detecting container is comprised of a first substrate provided with a recessed part and a second light-transmitting substrate arranged in such a way as to cover the recessed part. The first substrate and the second substrate are glued to each other by the adhesive, and a light shielding layer is provided for at least one surface of the second substrate corresponding to a surface at least provided with the adhesive in the fluorescence detecting container.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a container used for detecting a fluorescent substance or a sample labeled with the fluorescent substance.

[0002]

2. Description of the Related Art At present, there are a wide variety of shapes of containers used for fluorescence detection. For example, a cubic container simply made of a non-fluorescent material like a cell of a fluorescence spectrometer, a 96-well multititer plate, etc. Well-shaped container, DNA capillary disclosed in JP-A-11-75812, and JP-A-10-75812
And a tubular container or the like in which a small-capacity chamber or the like provided in the reactor of 185922 is formed.

[0003] These containers for fluorescence detection have been manufactured using materials suitable for fluorescence detection with as little autofluorescence as possible. For example, when fluorescence of a sample is detected using a fluorescence microscope, a slide glass or a cover glass made of non-fluorescent glass is used so that the fluorescence detection of the sample is not affected. When fluorescence is detected using a 96-well multititer plate, it is necessary to select a plastic that is a material of the plate that does not affect the detection of the fluorescence wavelength emitted by the sample.

As described above, the container used for the fluorescence detection uses a non-fluorescent material, or even if it emits fluorescence, it is limited to a material that emits fluorescence of a wavelength different from the detection fluorescence to some extent. I have. Alternatively, the structure or the arrangement is such that when the inside of the container is observed from the optical projection surface to detect the fluorescence, there is no material that emits fluorescence in the detection range.

[0005] On the other hand, in the prior art, in order to eliminate fluorescence from the container, which causes noise in fluorescence detection, the material and processing method of the container are greatly restricted. For example, containers with small volumes such as Affymetrix's GeneChip,
An example of the electrophoresis capillary of Shimadzu Corporation is a fluorescence detection container in which two or more members are joined. In such a container using two or more members, it is necessary to join each member. These are bonded by using an adhesive or bonded by an anodic bonding method.

[0006] The anodic bonding method is a method in which materials to be bonded are directly bonded without requiring any intervention. Therefore, if the material to be joined does not generate fluorescence, there is no problem of fluorescence even after joining. On the other hand, Affymetrix's GeneChip using an adhesive has a structure in which the adhesive does not extend to the extent that fluorescence is observed.

However, when using the anodic bonding method, it is necessary to heat to 500 ° C. and simultaneously apply a high voltage of 1 kV. Further, in order to achieve a structure in which the fluorescent adhesive does not reach the detection area, the container itself needs to be large. It is also possible to provide a filter on the detection optical system side to block the fluorescence generated from the container or, if laser excitation light is used, to limit the scanning range to avoid unnecessary fluorescence. However, such means causes the optical system to be complicated and the operation of the detection device to be complicated.

[0008]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to minimize the design restrictions on the structure of a container and a detection system, and to make it possible to freely design a container.
Also, it is an object of the present invention to provide a technique that allows a desired adhesive to be used freely without being restricted by the presence or absence of the fluorescence when the adhesive is used.

[0009]

In order to solve the above-mentioned problems, the present invention comprises a first substrate having a concave portion and a light-transmitting second substrate disposed to cover the concave portion. The first substrate and the second substrate are bonded with an adhesive, and a metal layer is provided on at least a surface of the second substrate corresponding to a surface provided with the adhesive. The present invention provides a fluorescence detection container characterized in that:

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Apparatus Hereinafter, using a partial cross-sectional view of a preferred example of the present invention,
The present invention will be described. The fluorescence detection container 1 is composed of a first substrate 2 having a concave portion 3 for holding a test sample or a reaction solution therein, and a light transmissive member adhered to the first substrate 2 by an adhesive layer 5. And at least one of the second substrates corresponding to the surface provided with the adhesive.
And a light shielding layer 6 made of metal such as chromium and aluminum provided on the surface. The light-shielding layer 6 is formed of the adhesive layer 5
It is arranged in order to shield the auto-fluorescence generated from, and to prevent the adhesive layer 5 from being irradiated with excitation light. Here, as an example, it is assumed that the fluorescence detection and the excitation light irradiation are both performed from above the second substrate.

The second base is provided with a sample inlet / outlet 7 for adding or discharging a sample to / from the recess 3. In this example, the sample inlet / outlet 7 is formed as two inlets and outlets, but may be one common inlet / outlet.
The size, form, and direction of perforation are not particularly limited as long as it is appropriate for adding and discharging the sample to and from the recess 3.

The first base 2 and the second base 4 may be made of the same material or different materials. These may be any of glass, silicon, metal, resin, and the like, but are desirably materials having no auto-fluorescence.
The size of the substrate is, for example, 1 cm to 30 cm in length and 1 c in width.
m to 30 cm and a depth of 0.01 cm to 0.8 cm.

[0013] Even when a light-transmitting member is joined to a container made of a fluorescent material, the light-transmitting member is provided with a light-blocking member even if the container is formed by a method other than bonding. Fluorescent light can be blocked.

The recess 3 can be formed by forming a recess in the first base. The concave portion can be formed by means known per se depending on the material. The capacity of the recess 3 is 1 μL to 3 mL, and can be changed according to the application.

The adhesive 5 may be any commonly used adhesive, for example, an epoxy adhesive can be suitably used.

As described above, the light shielding layer 6 is formed of a thin metal layer such as chromium and aluminum.

FIG. 2 is a sectional view of a preferred embodiment of the present invention shown in FIG. As shown in FIG. 2, in this example,
A light-blocking layer 6 is arranged on the top layer of the device. Further, the light-shielding layer 6 may extend above the recess 3 by a distance L shown in FIG. 2 in order to completely block auto-fluorescence from the adhesive layer 5. Further, as shown in FIG. 3, the light-shielding layer 6 is
And may be arranged directly above (FIG. 3). Furthermore, it may be arranged on the side surface of the first base and the second base. Here, “corresponding to the surface to which the adhesive is applied” refers to a surface capable of preventing the irradiation of the adhesive layer 5 with excitation light or the detection of fluorescence. The surface of the substrate may be only one surface or a plurality of surfaces. Further, the surface on which the light shielding layer 6 is arranged does not necessarily need to be parallel to the surface on which the adhesive layer 5 is applied.

Further, as shown in FIG. 3, the light-transmitting window 8 is formed in a portion where the light-shielding layer 6 is not applied, so that the light-transmitting window 8 is maintained for observing the sample by fluorescence detection.

According to the present invention, by combining a member that emits fluorescent light and a light-shielding member, it is possible to emit fluorescent light of a desired shape when detecting fluorescent light. That is, as shown in FIG. 4, it is possible to form a desired slit in the light shielding layer 6. With this slit, it is possible to observe the fluorescence from the adhesive at the position of the slit when detecting the fluorescence. By utilizing this, it is possible to easily confirm the position of the fluorescent site of the detected sample. It is also possible to identify a fluorescent site. Further, it can be used as a standard for the fluorescence intensity.

2. Manufacturing Method Next, an example of a manufacturing method when a silicon substrate is used as the first base and quartz glass is used as the second base will be described with reference to FIG.

First, an example of a method for forming a light-shielding layer on the second base will be described. First, a chrome film 11 is formed on a synthetic quartz glass 10 having a thickness of 0.5 mm by an appropriate thickness, for example, 300 mm.
It is formed by sputtering at 0 Å (FIG. 5-I-1). Next, the resist 12 is uniformly applied by a spin coater (FIG. 5-I-2). This is masked, exposed by an exposure machine, and developed to form a resist pattern (FIG. 5-I-3). According to the resist pattern, the chromium film is dissolved with hydrochloric acid (FIG. 5-I-4). Finally, the resist is oxidized and removed by plasma asher (Fig. 5-I-
Five).

Next, an example of a method for forming a concave portion in the first base will be described. First, a 0.5 mm thick silicon wafer 13
A silicon nitride film 14 having an appropriate thickness, for example, 5000 angstroms, is formed on the surface (FIG. 5-II-1). Next, the resist 15 is uniformly applied by a spin coater (see FIG.
5-II-2). Exposure is performed using a mask and an exposure machine, followed by development to form a resist pattern (FIG. 5-II-3). An appropriate remover for removing the nitride film not protected by the resist, for example, RI
E to remove (Fig. 5-II-4). Further, silicon is etched with a mixed solution of boiling nitric acid and acetic acid to form a concave portion having a depth of 0.1 mm (FIG. 5-II-5). The resist is oxidized and removed by plasma asher (Fig. 5-II-6). The adhesive layer 16 is printed by a screen printing machine (FIG. 5-II-7).

The first substrate and the second substrate obtained as described above are adhered together under a stereoscopic microscope and joined at 80 ° C. for 2 hours (FIG. 5-III). The bonding can be performed as shown in FIG.
As shown in FIG. 5A, the light shielding layer 11 may be disposed on the outside, or may be disposed on the inside as shown in FIG. 5B.

The above-described manufacturing method is an example for manufacturing the fluorescence detecting container of the present invention, and therefore, it can be manufactured by another method known per se.

For example, when the epoxy adhesive is excited by light of about 550 nm, which is an excitation light for a fluorescent substance such as Cy3, fluorescence in a wavelength range for detecting the fluorescence of Cy3 is strongly generated. For this reason, in the fluorescence detection container as shown in FIG. 1, when there is no light blocking member, the fluorescence of the epoxy adhesive at the peripheral portion of the container is strongly detected simultaneously with the fluorescence of the sample in the container. In such a case, the gain of the photomultiplier tube for detecting the fluorescence cannot be increased, but by arranging the light shielding member as in the fluorescence detection container of the present invention, the fluorescence derived from the epoxy adhesive is blocked. Further, in the detection device configured to apply the excitation light from the light transmissive member side, the excitation light is also blocked, so that there is an advantage that the epoxy adhesive does not emit any fluorescence.

In order to make the shape of the concave portion 3 of the fluorescence detection container or the like of the present invention shown in FIG. 1 suitable for electrophoresis, a groove having a width and depth of about 50 μm and a length of about 10 cm, It is also possible to use a capillary part of a capillary electrophoresis apparatus. This groove can be formed by sand blasting and processing. If you want to use for electrophoresis,
It is preferable to use synthetic quartz glass for both the first substrate and the second substrate. In addition, when performing electrophoresis, insulating properties are required. Therefore, as shown in FIG. 2, the chromium thin film is arranged so as not to come into contact with a member constituting the first base. However, when a material having no conductivity is used for the light shielding member, the arrangement of the light shielding layer does not necessarily need to be as shown in FIG.

The fields in which the fluorescence detection container of the present invention can be used include electrophoresis, polymerase chain reaction (PCR)
Method), fluorescence in situ hybridization (FISH), immunoreaction, and any other method used as an indicator for detecting a fluorescent substance.

[0028]

According to the fluorescence detection container of the present invention, there is provided a container which can be freely designed while minimizing design restrictions on the structure of the container and the detection system. Also, regarding the use of an adhesive in the production of the container, there is provided a technique that allows a desired adhesive to be used freely at a desired location without being bound by the presence or absence of fluorescence derived from the adhesive.

[Brief description of the drawings]

FIG. 1 is a partial cross-sectional view of a preferred example of a fluorescence detection container of the present invention.

FIG. 2 is a sectional view of a preferred example shown in FIG. 1;

FIG. 3 is a sectional view of a further preferred example of the present invention.

FIG. 4 is a plan view of a further preferred example of the present invention.

FIG. 5 is a scheme diagram showing an example of a method for manufacturing a fluorescence detection container of the present invention.

[Explanation of symbols]

1. Fluorescence detection container 2. First substrate 3. Reaction section
4. Second substrate 5. Adhesive layer 6. Shading layer
7. 7. Sample inlet / outlet Light transmission window

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Claims (2)

[Claims] 1. A fluorescence detection container comprising: a first substrate having a concave portion; and a light-transmissive second substrate disposed to cover the concave portion, wherein the first substrate and the second substrate are A fluorescence detection container, wherein a substrate is adhered with an adhesive, and a light-shielding layer is provided on at least one surface of a second substrate corresponding to at least the surface provided with the adhesive. 2. The fluorescence detecting container according to claim 1, wherein said light shielding layer is made of one of chromium and aluminum.