JPH08210983A - Method and device for detecting chemiluminescence - Google Patents

Abstract

PURPOSE: To provide a method and device for detecting chemiluminescence in which information related to polymer such as genic information can be efficiently and precisely obtained. CONSTITUTION: A polymer selectively labeled by a labeling material causing a chemiluminescence by the contact with a chemiluminescence material is housed in a visual ray transmitting housing member 4, an accumulating phosphor sheet 7 is brought into contact with the housing member 4 to expose the accumulating phosphor sheet 7 by the chemiluminescence caused by the contact of the polymer labeled by the labeling material with the chemiluminescence material, and the radiation energy accumulated in the accumulating phosphor sheet 7 is released. Thereafter, the accumulating phosphor sheet 7 is scanned by laser beams and excited, and the light caused by the excitation is photoelectrically detected by a light detector, whereby the image data is read.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemiluminescence detection method and apparatus for obtaining positional information of a labeling substance in a polymer, and more specifically to chemiluminescence using a stimulable phosphor. The present invention relates to a detection method and device.

[0002]

2. Description of the Related Art A fixed polymer such as a protein or a nucleic acid sequence is selectively labeled with a labeling substance which is brought into contact with a chemiluminescent substance to cause chemiluminescence, and is selectively labeled with the labeling substance. By contacting a macromolecule with a chemiluminescent substance, and detecting chemiluminescence in the visible light wavelength range caused by the contact between the chemiluminescent substance and the labeling substance, information about the macromolecule such as gene information can be obtained. Chemiluminescent detection methods are known. This chemiluminescence detection method
Determination of nucleotide sequences of nucleic acids such as DNA and RNA by electrophoresis, screening of genes by electrophoresis and colony hybridization, generation of reference data of genes by dot blotting, simple detection of gene amount and expression level, It is widely used for protein separation, identification, and evaluation of molecular weight and properties. Conventionally, such chemiluminescence is detected by exposing and developing a photographic film by chemiluminescence emitted from a polymer.
This was done by detecting a visible image. However, since chemiluminescence generated by contact between a chemiluminescent substance and a labeling substance is weak light, it is necessary to use a high-sensitivity photographic film with a high γ value in order to reliably detect it with a photographic film. However, when using a high-sensitivity photographic film with a high γ value, it is difficult to make sure that the exposure is done using the straight line portion of the characteristic curve, and there are many exposure mistakes. There has been a problem that it is necessary to repeatedly expose the film by changing the sensitivity of the film or the exposure time, and the chemical treatment of development is indispensable, which makes the operation troublesome. Furthermore, it is difficult to detect information on macromolecules such as genetic information with high accuracy by visual analysis of the image obtained by exposing the photographic film. There is a problem in that it is necessary to scan an image with light, convert it into a digital signal, perform digital image processing, and analyze the image, which requires a complicated operation.

Further, when it is irradiated with light in the visible light wavelength range, its energy is accumulated, and when it is subsequently irradiated with electromagnetic waves such as visible light, ultraviolet rays and infrared rays, it is excited and accumulated. Generated by contact between a chemiluminescent substance and a labeling substance using a stimulable phosphor sheet having a stimulable phosphor layer containing a stimulable phosphor that emits light in proportion to the energy of light in the light wavelength region. The chemiluminescence is stored and recorded in the stimulable phosphor sheet, and then the stimulable phosphor sheet is scanned using electromagnetic waves such as visible light, ultraviolet rays, infrared rays, etc. A chemiluminescence detection method is known in which energy is emitted, the emitted light is converted into a digital signal, and the obtained digital signal is image-processed to obtain information on macromolecules such as genetic information (for example, And JP-A-4-232864 discloses.). According to the chemiluminescence detection method using such a stimulable phosphor sheet, the stimulable phosphor emits light proportional to the energy of light in the irradiated visible light wavelength region when excited by electromagnetic waves. Compared with the case of using a photographic film, it is simple in that there are few exposure errors and there is no need for chemical treatment such as development. Furthermore, information on macromolecules such as genetic information is digital. Since it can be obtained in the form of a signal, there is an advantage that necessary image processing can be performed to accurately analyze information on the polymer.

[0004]

However, in such a chemiluminescence detection method, the stimulable phosphor has the property of accumulating it when irradiated with light in the visible light wavelength range. It is always necessary to block visible light, which is inconvenient to handle. On the other hand, for the same purpose as in the chemiluminescence detection method, a radiolabel is attached to a tissue of an organism and / or a substance derived from the organism, and the tissue of the organism and / or a substance derived from the organism to which the radiolabel is attached An autoradiographic measurement method is known in which the position information of a radiolabeled substance in a medium containing is absorbed, and the energy of radiation is absorbed and accumulated, and then electromagnetic waves such as visible light and infrared light are used. When excited, the stimulable phosphor layer containing a stimulable phosphor having a characteristic of emitting a stimulable amount of light in accordance with the amount of irradiated radiation energy is used to label a stimulable phosphor sheet with a radioactive label. An autoradiography measuring method used as a detection material for obtaining positional information of a substance has been proposed (for example, Japanese Patent Publication No. 1-60784 and Japanese Patent Publication No. 1).
No. -60782, Japanese Patent Publication No. 4-3952, etc. ).

Since this autoradiographic measuring method is used for the same purpose as the chemiluminescence detecting method, the same stimulable phosphor sheet is used, depending on the object, or the autoradiographic method. Using the measurement method,
Alternatively, it is desirable for users to be able to obtain information about macromolecules such as genetic information using chemiluminescent detection methods. However, in the chemiluminescence detection method, it is necessary to detect chemiluminescence in the visible light wavelength range caused by contact between the chemiluminescent substance and the labeling substance, while in the autoradiography measurement method, radiation is detected. Therefore, the same stimulable phosphor sheet could not be used for both chemiluminescence detection method and autoradiographic measurement method.

[0006]

An object of the present invention is to efficiently and efficiently use a stimulable phosphor sheet that is easy to handle and can be used in both chemiluminescence detection methods and autoradiography measurement methods. Another object of the present invention is to provide a chemiluminescence detection method capable of accurately obtaining information on macromolecules such as gene information. A second object of the present invention is to use a stimulable phosphor sheet that is easy to handle and can be used for both chemiluminescence detection methods and autoradiography measurement methods, and is efficient and accurate for gene analysis. It is an object of the present invention to provide a chemiluminescence detection device capable of obtaining information about a polymer such as information.

[0007]

According to the first aspect of the present invention, a macromolecule is selectively labeled with a labeling substance that comes into contact with a chemiluminescent substance to generate chemiluminescence. By contacting the polymer labeled with a substance and the chemiluminescent substance, chemiluminescence generated by contact between the polymer labeled with the labeling substance and the chemiluminescent substance, energy of radiation can be stored, Accumulation in which a stimulable phosphor layer is formed that includes a stimulable phosphor that is excited by light in the visible light wavelength range and emits the energy of the accumulated radiation as light. And a chemiluminescence detection method, which comprises exposing a fluorescent phosphor sheet to light. In a preferred embodiment of the first aspect of the present invention, the radiation is
It is composed of light having higher energy than light in the visible light wavelength range. In a further preferred embodiment of the first aspect of the present invention, the method for detecting chemiluminescence further comprises scanning the stimulable phosphor sheet exposed by chemiluminescence with electromagnetic waves to excite the stimulable phosphor sheet. The step of photoelectrically converting the light emitted from the device to obtain image data is provided.

A second object of the present invention is, according to the second aspect of the present invention, a polymer selectively labeled with a labeling substance which is brought into contact with a chemiluminescent substance to cause chemiluminescence, and the chemiluminescent substance. Visible light-transmissive accommodating member that can be contacted with, and radiation energy that can store the energy of radiation and is excited by light in the visible wavelength range and emits the energy of the stored radiation as light. A stimulable phosphor layer containing a phosphor is formed, and a stimulable phosphor sheet in which the energy of radiation is uniformly accumulated in advance, and the chemiluminescence generated by the contact between the labeling substance and the chemiluminescent substance cause accumulation. Exciting light irradiating means for irradiating excitation light to the stimulable phosphor sheet that has emitted the energy of the radiation as light, and emitted from the stimulable phosphor sheet by irradiating the excitation light. The light is accomplished by chemiluminescent detection apparatus having a detection means for photoelectrically detecting. In a preferred embodiment of the second aspect of the present invention, the radiation is
It is composed of light having higher energy than light in the visible light wavelength range. In a further preferred aspect of the second aspect of the present invention, the excitation light irradiating means is configured to scan the stimulable phosphor sheet with an electromagnetic wave to excite it.

In a further preferred embodiment of the second invention
In addition, the chemiluminescence detection device further comprises:
Equipped with a pre-exposure unit that uniformly irradiates radiation on the photo sheet
I am. In the present invention, radiation means X-ray, α
It contains rays, β rays, γ rays, electron rays and ultraviolet rays. Book
Formed into a stimulable phosphor sheet that can be used in the invention
As the stimulable phosphor contained in the stimulable phosphor layer,
It can store the energy of rays and
Energy of the accumulated radiation that is excited by
As long as it can be released as
Not of. Specifically, for example, JP-A-55-12
Alkaline earth metal fluoride halogen disclosed in Japanese Patent No. 145
Halide phosphor (Ba_1-x,M²⁺ _x) FX: yA (here
To M²⁺Consists of Mg, Ca, Sr, Zn and Cd
At least one alkaline earth metal element selected from the group
Element, X is a small number selected from the group consisting of Cl, Br and I
At least one kind of halogen, A is Eu, Tb, Ce, T
Consists of m, Dy, Pr, He, Nd, Yb and Er
At least one trivalent metal element selected from the group, x is 0
≦ x ≦ 0.6, y is 0 ≦ y ≦ 0.2. ), JP
Alkaline earth metal disclosed in JP-A-2-276997
Fluorohalide-based phosphor SrFX: Z (where X is
At least selected from the group consisting of Cl, Br and I
One kind of halogen, Z is Eu or Ce. ), JP
With europium disclosed in Japanese Patent Publication No. 59-56479
Active composite halogen-based phosphor BaFX.xNaX ': aE
u²⁺(Here, X and X'are both Cl, Br, and
And at least one halogen selected from the group consisting of
X is 0 <x ≦ 2, and a is 0 <a ≦ 0.2.
It ), Disclosed in JP-A-58-69281.
M, which is a trivalent metal oxyhalogen-based phosphor activated by lithium
OX: xCe (where M is Pr, Nd, Pm, Sm,
Eu, Tb, Dy, He, Er, Tm, Yb and Bi
At least one trivalent metal element selected from the group consisting of
Prime, X is one or both of Br and I, x
Is 0 <x <0.1. ), JP-A-60-1011
No. 79 and No. 60-90288.
Cerium-activated rare earth oxyhalogen-based phosphor
LnOX: xCe (where Ln is Y, La, Gd and
And at least one rare earth element selected from the group consisting of Lu
The element, X, is selected from the group consisting of Cl, Br and I
At least one halogen, x is 0 <x ≦ 0.1
It ) And JP-A-59-75200.
Europium-activated composite halide phosphor M^IIFX
aM^IX'bM^'IIX^'' ₂・ CM^IIIX^''' ₃・ X
A: yEu²⁺(Here, M^IIIs Ba, Sr and Ca?
At least one alkaline earth gold selected from the group consisting of
Genus element, M^IIs composed of Li, Na, K, Rb and Cs
At least one alkali metal element selected from the group
M^IIIs at least selected from the group consisting of Be and Mg
Is also a divalent metal element, M^IIIIs Al, Ga, In and
And at least one trivalent gold selected from the group consisting of Tl
Group elements, A is metal oxide, X is Cl, Br and I
At least one halogen selected from the group consisting of: X ',
X^''And X^'''Is a group consisting of F, Cl, Br and I
Is at least one halogen selected from
0 ≦ a ≦ 2 and b are 0 ≦ b ≦ 10^-2, C is 0 ≦ c ≦ 1
0 ^-2And a + b + c ≧ 10^-2And x is 0 <
When x ≦ 0.5, y is 0 <y ≦ 0.2. ) Is good
It can be used better.

In the present invention, the chemiluminescent substance is
If the chemiluminescent substance used in the chemiluminescent detection method,
It is not particularly limited and includes, for example, luminol, lucigenin, acridinium ester, AMPPD
(Adamantyl-1,2-dioxetane) and other dioxycetanes, "CDP-Star" manufactured by TROPIX
(Registered trademark), “CDP” and the like can be preferably used. In the present invention, the labeling substance that causes chemiluminescence upon contact with the chemiluminescent substance is not particularly limited as long as it is a chemiluminescent substance used in the chemiluminescence detection method, but it is not limited to a catalyst or an enzyme. Is preferably used.
Examples of catalysts include, for example, various metals and adenosine triphosphate, and examples of enzymes include lactate dehydrogenase, luciferase,
Examples include phosphatases such as peroxidase and alkaline phosphatase. Among these, enzymes,
Particularly, phosphatase is preferable, and alkaline phosphatase can be most preferably used. In the present invention, determination of the base sequence of nucleic acids such as DNA and RNA by electrophoresis, screening of genes by electrophoresis and colony hybridization, generation of reference data of genes by dot blotting, determination of gene amount and expression amount Target DN for easy detection
A labeling substance for labeling DNA or RNA complementary to A or RNA can be used, and examples of such a labeling substance include digoxigenin (DIG), avidin, streptavidin, biotin, and fluorescein. To be

[0011]

According to the first aspect of the present invention, a stimulable phosphor containing a stimulable phosphor capable of accumulating radiation energy and being excited by light in the visible wavelength region and capable of emitting the accumulated radiation energy as light. A stimulable phosphor sheet on which a stimulable phosphor layer has been formed and in which radiation energy has been uniformly accumulated is exposed by chemiluminescence generated by contact between a polymer labeled with a labeling substance and a chemiluminescent substance. Then, the stimulable phosphor is excited, and the stimulable phosphor emits the energy of radiation that has been accumulated in advance as light, and the accumulated energy is erased. Since the phosphor is recorded, it is not necessary to shield the stimulable phosphor sheet from visible light, and the stimulable phosphor sheet can be handled easily, efficiently and accurately, and the genetic information It is possible to obtain information on polymer such. Further, since the stimulable phosphor can store the energy of radiation, the stimulable phosphor sheet is
Not only the chemiluminescence detection method but also the autoradiography measurement method used for the same purpose can be used. Therefore, the user can select the chemiluminescence detection method or the autoradiography measurement method depending on the target. Information on macromolecules such as gene information can be obtained by determining the base sequence of nucleic acids such as DNA and RNA by electrophoresis, screening of genes by electrophoresis or colony hybridization, genes by dot blotting. Generation of reference data for
It is possible to efficiently perform simple detection of gene amount and expression amount, separation and identification of proteins, or evaluation of molecular weight and characteristics.

According to a further preferred embodiment of the first aspect of the present invention, the stimulable phosphor sheet exposed by chemiluminescence is further scanned and excited by electromagnetic waves to emit light emitted from the stimulable phosphor sheet. Since the image data is obtained by photoelectric conversion, the image data can be subjected to digital image processing and easily analyzed, with high accuracy.
It becomes possible to obtain information on macromolecules such as genetic information. According to the second aspect of the present invention, stimulable fluorescent light containing a stimulable phosphor capable of accumulating radiation energy, being excited by light in the visible light wavelength range, and being capable of releasing the energy of the accumulated radiation as light. A stimulable phosphor sheet on which a body layer has been formed and on which radiation energy has been evenly accumulated is exposed by chemiluminescence generated by contact between a polymer labeled with a labeling substance and a chemiluminescent substance, and stimulated. Chemiluminescence is recorded in the stimulable phosphor by exciting the stimulable phosphor and causing the stimulable phosphor to release the energy of the radiation that has been stored in advance as light and erase the stored energy. It is not necessary to shield the stimulable phosphor sheet from visible light because it is provided with an exposure means for controlling, and the stimulable phosphor sheet can be handled easily, efficiently and with good accuracy. , It is possible to obtain information on polymer such as genetic information.

Further, since the stimulable phosphor can store the energy of radiation, the stimulable phosphor sheet can be used not only for the chemiluminescence detection method but also for the autoradiographic measurement method used for the same purpose. Therefore, the user can use the chemiluminescence detection method or the autoradiography measurement method depending on the target.
It is possible to obtain the positional information of the labeling substance in the polymer, to determine the base sequence of nucleic acids such as DNA and RNA by the electrophoresis method, screen the gene by the electrophoresis method or colony hybridization, and detect the gene by the dot blotting. It is possible to efficiently perform reference data generation, simple detection of gene amount and expression amount, protein separation and identification, or evaluation of molecular weight and characteristics. According to a further preferred embodiment of the second aspect of the present invention, the stimulable phosphor sheet exposed by the exposure means is further scanned by electromagnetic waves to be excited, and light emitted from the stimulable phosphor sheet is photoelectrically converted. Since the image data is provided with a reading means, the image data can be subjected to digital image processing and easily analyzed, and it is possible to accurately obtain information on macromolecules such as gene information. Become.

[0014]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. FIG.
And FIG. 2 is a flow sheet showing the procedure of the chemiluminescence detection method according to the embodiment of the present invention. 1 and 2
Shows an example of a chemiluminescence detection method in the case where the gene amount is detected by dot blotting to generate gene reference data. In FIGS. 1 and 2, first, a dilution series 1 of 7 unlabeled DNAs is prepared. Each dilution series 1 is, for example, 10 ng
/ Microliter, 1ng / microliter, 100
pg / microliter, 10 pg / microliter, 1
It contains pg / microliter, 100 fg / microliter and 10 fg / microliter of DNA.
Then, using a pipette, each diluted solution of DNA is dropped on the nylon membrane filter 2 to form spots 3.

After the membrane filter 2 is dried, it is irradiated with ultraviolet rays to cross-link the DNA and the membrane filter 2 (UV crosslink), and the DNA is fixed on the membrane filter 2. After that, the membrane filter 2 on which the DNA is fixed is capable of transmitting visible light and
It is housed in a sealable plastic hybridization bag 4. Next, a hybridization solution is prepared and poured into the hybridization bag 4 for prehybridization. Meanwhile, a complementary DNA probe labeled with digoxigenin (DIG) -dNTP was prepared, added to the hybridization solution, and the hybridization solution for prehybridization was discharged from the hybridization bag 4, 4 and hybridize with the DNA immobilized on the membrane filter 2. In FIG. 1, 5
Indicates DNA hybridized with complementary DNA. In the present embodiment, since the gene amount is detected and the reference data of the gene is generated, all the DNA dropped on the membrane filter 2 is hybridized with the complementary DNA.

After completion of the hybridization, the membrane filter 2 is washed, and an anti-digoxigenin alkaline phosphatase labeled antibody solution is prepared and added to the hybridization bag 4. As a result, the complementary D labeled with digoxigenin (DIG)
DNA binding to NA and alkaline phosphatase
Are labeled. In FIG. 2, 6 indicates a complementary DNA labeled with alkaline phosphatase. In this embodiment, the membrane filter 2
Complementary DNA hybridized with all the DNA dropped above is labeled with alkaline phosphatase. Next, after discharging the anti-digoxigenin alkaline phosphatase-labeled antibody solution from the hybridization bag 4, the AMPPD (adamantyl-1,2-dioxetane) solution is added into the hybridization bag 4. When AMPPD contacts alkaline phosphatase, it decomposes and emits chemiluminescence. Therefore, chemiluminescence is generated from the DNA labeled with alkaline phosphatase. On the other hand, the surface of the stimulable phosphor sheet 7 having a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor capable of accumulating radiation energy is uniformly irradiated with low energy X-rays. Then, low-energy X-ray energy is uniformly accumulated in the stimulable phosphor layer. Here, the wavelength of chemiluminescence generated by the decomposition of AMPPD is about 470 nm, while the peak of the excitation wavelength of the barium fluorobromide-based stimulable phosphor used in the autoradiography measurement method is Since it is on the long wavelength side, in order to efficiently detect chemiluminescence, a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor that is used in an autoradiography measurement method is formed. Among the stimulable phosphor sheets 7 thus prepared, it is preferable to use one in which the peak of the excitation wavelength of the stimulable phosphor is on the shorter wavelength side. When a barium fluorobromide-based stimulable phosphor is used as the stimulable phosphor, the peak of the excitation wavelength can be shifted to the short wavelength side by increasing the amount of Br.

Next, the hybridization bag 4
After discharging a part of the AMPPD solution, the hybridization bag 4 is used to increase the decomposition rate of AMPPD.
Then, the stimulable phosphor sheet 7 on which the stimulable phosphor layer in which low-energy X-ray energy is evenly accumulated is formed, and the hybridization bag 4 are placed on a surface in a dark room. Contact. Here, in the present embodiment, a barium fluorobromide-based stimulable phosphor capable of accumulating radiation energy and being excited by an electromagnetic wave in the visible light wavelength range is used.
When the stimulable phosphor layer formed on the stimulable phosphor sheet 7 is exposed by the chemiluminescence generated by the decomposition of AMPPD, the exposed portion of the stimulable phosphor layer is exposed to the accumulated X-rays. Energy is released and erased. After a lapse of a predetermined time, the stimulable phosphor sheet 7 is separated from the hybridization bag 4. Thus, in the stimulable phosphor layer of the stimulable phosphor sheet 7 exposed by chemiluminescence, the energy of X-rays is uniformly accumulated only in the part which is not exposed by chemiluminescence. In the exposed portion, the X-ray energy accumulated in advance is released and erased according to the intensity of chemiluminescence.
In this embodiment, an image corresponding to the DNA concentration of each spot 2 is accumulated and recorded at the position of the stimulable phosphor layer corresponding to each spot 2.

FIG. 3 thus shows that the images have been accumulated,
FIG. 3 is a schematic perspective view showing an example of an image data reading device and an image processing device for reading image data from a stimulable phosphor sheet 7 on which a recorded stimulable phosphor layer is formed.
The image data reading device uses the stimulable phosphor sheet 7
The laser light 10 is configured to scan, excite, and generate stimulated light. The laser light 10 is generated by the laser light source 11 and passes through the filter 12 to cut a portion of the wavelength region corresponding to the wavelength region of the photostimulable light generated from the stimulable phosphor sheet 7 by the excitation by the laser light 10. To be done. Then, the laser light 10
The beam diameter is accurately adjusted by the beam expander 13 and enters the optical deflector 14 such as a galvanometer mirror. The laser beam 2 deflected by the optical deflector 14 is f
The light is reflected by the plane reflecting mirror 16 via the θ lens 15 and is one-dimensionally incident on the stimulable phosphor sheet 7.
The fθ lens 15 ensures that when the stimulable phosphor sheet 7 is scanned by the laser light 10, the scanning is always performed at a uniform beam velocity. In synchronization with the scanning by the laser light 10 as described above, the stimulable phosphor sheet 7 is moved in the direction of arrow A in FIG.
The entire surface is scanned by the laser light 10.

When the stimulable phosphor sheet 7 is irradiated with the laser beam 10, the stimulable phosphor sheet 7 emits a stimulable amount of light in proportion to the energy of the accumulated X-rays, and the stimulable emitted light has a light guiding property. It is incident on the sheet 17. The light guide sheet 17 has a light-receiving end portion which is linear and is arranged in proximity to face the scanning line on the stimulable phosphor sheet 7, and the emission end portion which has an annular shape. , A photomultiplier or other photoelectric conversion type photodetector 18 is connected to the light receiving surface. The light guide sheet 17 is made by processing a transparent thermoplastic resin sheet such as an acrylic synthetic resin, and the light incident from the light receiving end repeats total reflection on the inner surface of the light receiving end while the light exiting end is emitted. The shape is determined so as to be transmitted to the light receiving surface of the photodetector 18 through the section. Therefore, the photostimulable light emitted from the stimulable phosphor sheet 7 in response to the irradiation of the laser light 10 is incident on the light guide sheet 17, and inside the light guide sheet 17, while repeating total reflection, through the emission end portion, The light is received by the photodetector 18. A filter is attached to the light receiving surface of the photodetector 18, which transmits only the light in the wavelength region of the photostimulable light emitted from the stimulable phosphor sheet 7 and cuts the light in the wavelength region of the laser light 10. Cage, photodetector 18
Is configured to photoelectrically detect only the stimulated emission emitted from the stimulable phosphor sheet 7.

The photostimulable light photoelectrically detected by the photodetector 18 is converted into an electric signal, amplified by an amplifier 19 having a predetermined amplification factor into an electric signal of a predetermined level, and then A / D. It is input to the converter 20. The electrical signal is
In the A / D converter 20, it is converted into a digital signal with a scale factor suitable for the signal fluctuation width, input to the image processing device 21, stored in an image data memory (not shown), and if necessary, After performing predetermined digital image processing, it is displayed as an image on the screen of the CRT 22. According to this embodiment, a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor that does not accumulate visible light and can be used in an autoradiography measurement method is uniformly dispersed by low-energy X-rays. X-ray energy is accumulated evenly on the photostimulable phosphor layer by chemiluminescence generated by decomposition of AMPPD.
An image corresponding to the DNA concentration of each spot 2 is accumulated in the stimulable phosphor layer by releasing and erasing the linear energy, and then the stimulable fluorescence of the stimulable phosphor 7 is accumulated by the laser light 2. The body layer is scanned to generate photostimulated light, which is photoelectrically detected by the photodetector 18, converted into a digital signal by the A / D converter 20, and stored in the image data memory, If necessary, predetermined digital image processing is performed and reproduced on the screen of the CRT 22, so that the stimulable phosphor 7 does not always need to be shielded from visible light, which is convenient for handling. , The stimulable phosphor 7 can be shared with an autoradiographic measurement method used for the same purpose as the chemiluminescence detection method,
Furthermore, since it can be easily converted into a digital signal, chemiluminescence can be detected extremely efficiently and accurately. Furthermore, in this embodiment, since the stimulable phosphor sheet 7 having the stimulable phosphor layer containing the bromine-rich barium fluorobromide stimulable phosphor is used, chemiluminescence is more efficiently emitted. Can be detected.

FIG. 4 and FIG. 5 are flow sheets showing the procedure of the chemiluminescence detection method according to another embodiment of the present invention. 4 and 5 show a method for determining a DNA sequence by the Southern blotting method. In FIGS. 4 and 5, first, a DNA containing a target gene
The fragments 30 are electrophoresed on the agarose gel support medium 31 to be separated and developed. Then
As is well known, the DNA fragment 30 separated and developed on the agarose gel support medium 31 is denatured by alkali treatment, and the agarose gel support medium 31 and the nylon membrane filter 32 are allowed to stand for a predetermined time. Membrane contact 3
The denatured DNA fragment is transferred onto 2. Then, the membrane filter 32 is dried and irradiated with ultraviolet rays to cross-link the DNA and the membrane filter 32 (UV cross-link), and the DNA is fixed on the membrane filter 32.

Thereafter, the membrane filter 32, on which the DNA is fixed, is housed in a plastic hybridization bag 34 which is transparent to visible light and can be sealed. Next, a hybridization solution is prepared and poured into the hybridization bag 34 for prehybridization. On the other hand, a DNA probe complementary to the DNA containing the gene of interest labeled with digoxigenin (DIG) -dNTP is prepared and added to the hybridization solution, and the hybridization solution for prehybridization is added to the hybridization bag 34. After being discharged from the
The DNA immobilized on the membrane filter 32 is hybridized. In FIG. 4, 35 is a complementary DN
DN containing a gene of interest hybridized with A
A is shown. After the hybridization is completed in this way, the membrane filter 32 is washed, and an anti-digoxigenin alkaline phosphatase-labeled antibody solution is prepared, and the hybridization bag 34 is prepared.
Add in As a result, the complementary DNA labeled with digoxigenin (DIG) and alkaline phosphatase bind to each other to label the DNA. In FIG. 1, 36 indicates a complementary DNA labeled with alkaline phosphatase.

Next, the anti-digoxigenin alkaline phosphatase labeled antibody solution is discharged from the hybridization bag 34, and then the AMPPD solution is added into the hybridization bag 34. As a result, chemiluminescence is generated from the position where the DNA labeled with alkaline phosphatase is present. On the other hand, the surface of the stimulable phosphor sheet 37 on which the stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor capable of accumulating radiation energy is formed is uniformly irradiated with ultraviolet rays, Energy is accumulated in the stimulable phosphor layer. Here, the wavelength of the chemiluminescence generated by the decomposition of AMPPD is about 470 nm, while the peak of the excitation wavelength of the barium fluorobromide stimulable phosphor used in the autoradiography measurement method is Since it is on the long wavelength side, in order to efficiently detect chemiluminescence, a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor that is used in an autoradiography measurement method is formed. Of the stimulable phosphor sheet 37 thus prepared, it is preferable to use one in which the peak of the excitation wavelength of the stimulable phosphor is on the shorter wavelength side. When a barium fluorobromide-based stimulable phosphor is used as the stimulable phosphor, by increasing the amount of Br,
The peak of the excitation wavelength can be shifted to the short wavelength side.

Next, the hybridization bag 3
4, after discharging a part of the AMPPD solution, the hybridization bag 34 was heated to increase the decomposition rate of AMPPD, thus forming a stimulable phosphor layer in which the energy of ultraviolet rays was uniformly accumulated. The stimulable phosphor sheet 37 and the hybridization bag 34 are
Make surface contact in a dark room. As a result, the energy of ultraviolet rays accumulated in the portion of the stimulable phosphor layer exposed by the chemiluminescence generated by the decomposition of AMPPD is released and erased, and the image of DNA is transferred to the stimulable phosphor layer. Will be recorded. After a lapse of a predetermined time, the stimulable phosphor sheet 37
Is separated from the hybridization bag 34. Thus, the stimulable phosphor sheet 3 exposed by chemiluminescence
In the stimulable phosphor layer of 7, the ultraviolet energy is uniformly accumulated only in the part which is not exposed by the chemiluminescence, while the part which is exposed by the chemiluminescence, depending on the intensity of the chemiluminescence, The energy of ultraviolet light that has been accumulated in advance is released and erased, and the image of DNA is recorded. In this way, the image of the DNA recorded on the stimulable phosphor layer of the stimulable phosphor sheet 37 is read as a digital signal by the image data reading device shown in FIG. 3 and sent to the image processing device 21, It is stored in the image data memory, subjected to digital image processing as necessary, and displayed on the screen of the CRT 22.
DNA based on the image displayed on the screen of CRT22
The sequence can be easily determined.

Also in this embodiment, similarly to the above-mentioned embodiment, it becomes possible to detect chemiluminescence extremely efficiently and accurately.

[0026]

EXAMPLES Examples will be given below in order to further clarify the effects of the present invention. Example First, a dilution series of 7 unlabeled DNAs was prepared.
10 ng / microliter, 1 ng / microliter, 100 pg / microliter, 10 pg /
Microliter, 1pg / microliter, 100fg
/ Microliter and 10 fg / microliter of DNA. Then, using a pipette, each diluted solution of DNA was dropped onto a 15 cm × 10 cm nylon membrane filter having a pore diameter of 0.45 μm to form spots. After drying the membrane filter, irradiate it with UV light with a wavelength of about 265 nm, and
The NA and the membrane filter were cross-linked (UV cross-linked), and the DNA was immobilized on the membrane filter. Then, the membrane filter having the DNA immobilized thereon was moistened with sterilized water and housed in a plastic hybridization bag which was permeable to visible light and sealable.

Then, 30 ml of a hybridization solution consisting of 500 mM sodium phosphate buffer (pH 7.2), 7% SDS and 1 mM EDTA was added to the membrane containing the membrane filter on which the DNA was immobilized. Pre-hybridization was performed by pouring into a hybridization bag. On the other hand, a DNA probe was prepared as follows. 1 microgram of the DNA fragment was dissolved in 15 microliters of distilled water, placed in a microtube, sealed, put in boiling water, heated at 100 ° C. for 5 minutes, and denaturated. , The DNA fragment was single-stranded. The single-stranded DNA solution was rapidly cooled in ice water for 5 minutes, precipitated by a small centrifuge, and added with 2 microliters of the hexanucleotide mixture and 2 microliters of the dNTP labeling mixture,
Mixed well. Here, the dNTP labeling mixture contains digoxigenin-dNTP. In this mixture,
Add 1 microliter of Klenow enzyme and mix gently by pipetting at 37 ° C
The reaction was allowed to proceed for 2 hours.

The reaction was then stopped by adding 2 microliters of 2 mol EDTA. Furthermore, 2.5 microliters of 4 mol lithium chloride and 75 microliters of 100% ethanol are added, and it is -70 degreeC.
Then, it was left to stand for 1 hour to precipitate the precipitate. Then, it was centrifuged at 4 ° C. for 10 minutes at 15,000 rpm to collect the precipitate at the bottom of the tube. After gently aspirating the supernatant with a pipette, add 500 microliters of 70% ethanol and stir vigorously with a test tube mixer to confirm that the precipitate had peeled off from the bottom of the tube. Centrifugation was carried out at 4 ° C. for 5 minutes at 15,000 rpm, and the precipitate was collected again at the bottom of the tube. The supernatant was then gently pipetted off and air dried. Then, 50 microliters of TE buffer is added to prepare a DNA probe,
Stored at 20 ° C. A predetermined amount of the thus prepared DNA probe is put into a microtube, sealed, put into boiling water, and heated at 100 ° C. for 5 minutes to denaturate the DNA probe.
It was single-stranded.

The denatured DNA probe was placed in ice water for 5 minutes.
Quenched for a minute and allowed to settle. Then, cut off the corner of the hybridization bag and discard the pre-hybridization solution in the hybridization bag,
A solution prepared by adding a DNA probe to a concentration of 20 ng / ml in 10 ml of the hybridization solution which had been heated to 65 ° C. in advance,
Pour into a hybridization bag containing a membrane filter with DNA fixed, and seal.
Hybridization was carried out at 5 ° C. for 15 hours. After the hybridization was completed, the hybridization solution was discharged from the hybridization bag, and 40 mmol of sodium phosphate buffer solution (pH 7.2) that had been preheated and 1% S were added.
The washing solution consisting of DS was poured into the hybridization bag, the hybridization bag was sealed, and the membrane filter was washed while shaking vigorously. For cleaning, change the cleaning solution to a new cleaning solution and do 3
It was divided into two times and carried out for 20 minutes. After the washing is completed, the washing solution is discharged from the hybridization bag, and an anti-digoxigenin alkaline phosphatase-labeled antibody solution is prepared. Combine,
The DNA was labeled.

Then, the anti-digoxigenin alkaline phosphatase-labeled antibody solution was discharged from the hybridization bag, and A was placed in the hybridization bag.
MPPD solution was added. On the other hand, it was placed on the surface of the stimulable phosphor sheet on which the stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor was formed, at a position 50 cm away from the surface of the stimulable phosphor sheet. 8 kilovolts,
Low-energy X-rays were uniformly irradiated by an X-ray source of 150 McAmpere, and the low-energy X-rays were accumulated in the stimulable phosphor layer. On the other hand, after discharging a part of the AMPPD solution from the hybridization bag,
In order to increase the decomposition rate of MPPD, the hybridization bag was heated to 37 ° C. and hybridized with a stimulable phosphor sheet having a stimulable phosphor layer on which low-energy X-rays were uniformly accumulated. The bag was in surface contact in the dark for 4 hours. After that, FIG.
The surface of the stimulable phosphor sheet was scanned with the helium-neon laser light by using the image data reading device shown in FIG. 1 to photoelectrically detect the photostimulable light, which was read as a digital signal and reproduced on the CRT screen. By the way, 100 fg /
Microliters of DNA were detected.

The present invention is not limited to the above embodiments and examples, and various modifications can be made within the scope of the invention described in the claims, and these are also within the scope of the present invention. It goes without saying that it is included.
For example, in the above-mentioned embodiment, an example of detecting the gene amount and determining the nucleotide sequence of DNA by dot-blotting and electrophoretic method for generating reference data of the gene is shown. It is not limited to these, and can be used for the determination of nucleotide sequences of nucleic acids other than DNA by electrophoresis, screening of genes by electrophoresis or colony hybridization, separation and identification of proteins, or evaluation of molecular weight and characteristics. Can also be used. Therefore, the labeling substance and the chemiluminescent substance are not limited to those described in the above embodiment, and those suitable for each chemiluminescence detection method can be used. Further, in the above-mentioned Examples, an example of dot-blotting for generating gene reference data by detecting a gene amount is shown, but the present invention is also used for other chemiluminescence detection methods. can do. Furthermore, in the above-described embodiments and examples, as the stimulable phosphor, a barium fluorobromide-based phosphor is used, but as the stimulable phosphor, the energy of light having a wavelength other than the visible light wavelength range is used. Can be accumulated,
As long as it can be excited by light in the visible wavelength range, not only barium fluorobromide-based phosphors but also barium fluorohalide-based phosphors, other alkaline earth metal fluoride halide-based phosphors, or,
Europium-activated composite halogen-based phosphor, cerium-activated trivalent metal oxyhalogen-based phosphor, cerium-activated rare earth oxyhalogen-based phosphor, europium-activated composite halogen-based phosphor, etc. can also be used. .

In the above embodiment, the chemiluminescence information detected and converted into a digital signal is displayed as an image on the screen of the CRT 22, but it may be displayed on other display means. , Or photo film,
You may make it reproduce | regenerate on other recording media.

[0033]

EFFECTS OF THE INVENTION According to the present invention, a stimulable phosphor sheet which is easy to handle and can be used in both chemiluminescence detection methods and autoradiography measurement methods can be used efficiently and accurately. It is possible to provide a chemiluminescence detection method and device capable of obtaining information on macromolecules such as genetic information.

[Brief description of drawings]

FIG. 1 is a flow sheet showing a first half of a procedure of a chemiluminescence detection method according to an embodiment of the present invention.

FIG. 2 is a flow sheet showing the latter half of the procedure of the chemiluminescence detection method according to the embodiment of the present invention.

FIG. 3 is a schematic perspective view showing an example of an image data reading device and an image processing device.

FIG. 4 is a flow sheet showing the first half of the procedure of the chemiluminescence detection method according to another embodiment of the present invention.

FIG. 5 is a flow sheet showing the latter half of the procedure of the chemiluminescence detection method according to another embodiment of the present invention.

[Explanation of symbols]

1 Dilution series of DNA 2 Membrane filter 3 Spot 4 Hybridization bag 5 DNA 6 hybridized with complementary DNA 6 Complementary DNA labeled with alkaline phosphatase 7 Accumulative phosphor sheet 10 Laser light 11 Laser light source 12 Filter 13 Beam expander 14 Optical deflector 15 fθ lens 16 Planar reflecting mirror 17 Light guide sheet 18 Photodetector 19 Amplifier 20 A / D converter 21 Image processing device 22 CRT 30 DNA 31 Agarose gel support medium 32 Membrane Filter 34 Hybridization bag 35 DNA hybridized with complementary DNA 36 Complementary DNA 37 labeled with alkaline phosphatase 37 Accumulative phosphor sheet

Claims (7)

[Claims] 1. A macromolecule is selectively labeled with a labeling substance that is brought into contact with a chemiluminescent substance to generate chemiluminescence.
Energy of radiation can be accumulated by contacting the polymer labeled with the labeling substance with the chemiluminescent substance, and chemiluminescence generated by the contact between the polymer labeled with the labeling substance and the chemiluminescent substance. Then, a stimulable phosphor layer containing a stimulable phosphor that is excited by light in the visible light wavelength range and emits the energy of accumulated radiation as light is formed, and the energy of radiation is uniformly accumulated in advance. The method for detecting chemiluminescence, which comprises exposing the stimulable phosphor sheet. 2. The chemiluminescence detection method according to claim 1, wherein the radiation is light having higher energy than light in the visible light wavelength range. 3. Further, the stimulable phosphor sheet exposed by chemiluminescence is scanned by electromagnetic waves,
The chemiluminescence detection method according to claim 1, wherein the image data is obtained by photoelectrically converting light that is excited and emitted from the stimulable phosphor sheet. 4. A visible light-transmissive housing member for housing the polymer, which is selectively labeled with a labeling substance that comes into contact with a chemiluminescent substance to cause chemiluminescence, and the chemiluminescent substance so as to be contactable therewith. Can store the energy of radiation,
A stimulable phosphor layer containing a stimulable phosphor that is excited by the light in the visible light wavelength range and uses the accumulated energy of the radiation as light is formed, and the energy of the radiation is uniformly accumulated in advance. Irradiation of excitation light to the stimulable phosphor sheet, which has emitted the energy of the accumulated radiation as light by chemiluminescence generated by the contact between the stimulable phosphor sheet and the chemiluminescent substance. 2. A chemiluminescence detection device comprising: an excitation light irradiating unit that performs the above and a detection unit that photoelectrically detects the light emitted from the stimulable phosphor sheet by the irradiation of the excitation light. 5. The chemiluminescence detection device according to claim 4, wherein the radiation is light having a higher energy than light in the visible light wavelength region. 6. The chemiluminescence detection device according to claim 4, wherein the excitation light irradiation means is configured to scan the stimulable phosphor sheet with electromagnetic waves to excite it. . 7. The chemiluminescence detection device according to claim 4, further comprising a pre-exposure unit that uniformly irradiates the stimulable phosphor sheet with radiation.