JP2002340892A - Specimen material detection method using biochemical specific binding reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a detection method enabling detection of a specimen material such as an organism related substance or its duplicate via a biochemical specific reaction with high precision and sensitiveness. SOLUTION: In this detection method, a detection sheet, in which a probe molecule bondable with the specimen material is stuck to each of porous structural bodies in a composite material sheet constructed of the porous structural bodies arranged respectively in a plurality of areas sectioned by partition walls, and a stimulable phosphor sheet having a plurality of stimulable phosphor layers arranged independently of each other in the positions individually matching the porous structural body existing positions in the detection sheet are prepared, and using these sheets, a radiological image storage/reproduction method is carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for analyzing a sample substance such as a biological substance or a copy thereof,
In particular, a sample substance labeled with a radioactive substance is bound and immobilized on a porous detection sheet via a biochemical specific binding reaction,
The present invention relates to a method for detecting a sample substance using an operation of detecting the bound radioactive label using a stimulable phosphor sheet.

[0002]

2. Description of the Related Art In recent years, analytical tools called macroarrays and microarrays have been widely used for analyzing genes in the fields of biology and medicine.
All of these are analytical tools for detecting and analyzing nucleic acids such as DNA and RNA or fragments thereof or their duplicates by utilizing hybridization, which is a kind of biochemical specific binding reaction. The former macro array is
It consists of a porous sheet made of polyamide resin and the like, and the detection analysis using it is performed by fixing nucleic acid fragments (probe molecules) such as a large number of DNA fragments so as to entangle the pores of the porous sheet, The analysis is performed using a sample nucleic acid fragment labeled with a radioactive substance such as a radioisotope (RI) as an analysis target (target molecule). On the other hand, the latter microarray is composed of a solid support such as a surface-treated slide glass, and its detection analysis is usually performed by immobilizing probe molecules on the surface of the solid support and using a sample nucleic acid fragment labeled with a fluorescent substance as a target molecule. It is performed using. However, it seems that the use of the name of the macro array and the name of the micro array is generally not always strictly used.

[0003] Gene analysis using a macroarray is advantageous in that it can be easily performed using conventional autoradiography techniques.

[0004] Nuclei such as DNA using the above-mentioned macroarray
The analysis of the acid is usually performed by the following method. (1) First, for a plurality of types of nucleic acid fragments, a large number of single strands
Nucleic acid fragment (probe molecule; usually its base sequence is known
Is used, and an aqueous solution containing each is prepared.
On a macro array using a spotter at high density.
By sequentially spotting in the form of a rick
Attached so as to entangle with the pores at the spotting position on the porous sheet
To form a large number of dot-like probe molecule spots.
Let (2) Next, for the nucleic acid fragment sample to be analyzed,
Element (RI:³²P, ³³P, etc.)
A sample of the radioactively labeled single-stranded nucleic acid fragment is applied to the macroarray.
Phase contact (for example, radioactive
Immerse macroarray in aqueous solution of labeled nucleic acid fragment sample
The target molecule to be detected is
Hybridize and bind. That is, nucleic acids
Has a base sequence complementary to the probe molecule in the fragment sample
The target molecule complements the spot probe molecule.
Binding (hybridization). (3) Next, do not hybridize from the macro array.
The radioactively labeled nucleic acid fragment sample is washed away. (4) The washed and dried macroarray is
And autoradiography.
To detect radiation from radiolabeled target molecules
The hybridization of each probe molecule
Information on the binding of the target molecule by the
And intensity). (5) Base arrangement of probe molecule bound to target molecule
If the sequence is known, by using the principle of complementarity
The base sequence of at least a part of the target molecule is determined.
You. That is, using such a technology,
Expression, mutation, polymorphism, etc. for many genes simultaneously
Can be analyzed.

Heretofore, in general autoradiography of radiolabeled biological samples and biopolymers, instead of using a radiographic film, a radiation image conversion panel (imaging plate or stimulable fluorescent A radiation image recording / reproducing method using a body sheet) has been developed. This radiation image recording / reproducing method absorbs and accumulates a part of radiation energy when irradiated with radiation such as X-rays, and then accumulates the radiation energy when irradiated with electromagnetic waves (excitation light) such as visible light or infrared light. A stimulable phosphor (eg, a stimulable phosphor) having a property of exhibiting stimulable luminescence in accordance with the condition. After the radiation image information of the subject is once stored by irradiating the radiation transmitted from or emitted from the subject, the stimulable phosphor sheet is sequentially scanned with excitation light such as laser light to emit the stimulated emission light. The photostimulated emission light is read photoelectrically to obtain an electric image signal (digital signal), and the obtained digital signal is used as it is or after various signal processings are performed.
It consists of reproducing it by making it a visible image or storing it in an appropriate recording medium.

[0006] Autoradiography using the above-mentioned radiographic image recording / reproducing method using a stimulable phosphor sheet obtains a radiographic image with high sensitivity even when the radiation dose from a radioactive substance-labeled sample is extremely small. It is an important autoradiography technology at present because it has many advantages, such as image processing, and image information is obtained as a digital signal, which facilitates image processing and storage.

As a method for measuring radiation from a radiolabeled target molecule, a method using the above-mentioned stimulable phosphor sheet has already been proposed. For example, Human Mole
cular Genetics, 1999, Vol. 8, No. 9, 1715-1722,
A DNA fragment (probe molecule) is immobilized as a number of spots on the surface of the porous sheet, and this DNA fragment and a radiolabeled DNA fragment sample having complementarity are hybridized on the porous sheet. It describes that a target molecule can be detected by laminating a porous sheet and a stimulable phosphor sheet and performing autoradiography.

[0008]

In the above-described gene analysis using a porous sheet such as a macroarray, the autoradiography using a stimulable phosphor sheet is used to detect a radioactively labeled target molecule with high sensitivity. It is possible to do. However, when the probe molecule solution is spot-fixed, the attachment area of the probe molecules tends to spread in the plane direction of the porous sheet, so that the density of spots formed on the porous sheet (the number per unit area) is too high. Can not do it. In addition, when the attachment region of the probe molecule diffuses on the porous sheet, the immobilization region of the target molecule that hybridizes to the probe molecule similarly expands, so that the laminated exposure with the porous sheet carrying the radiolabeled target molecule is performed. Excited radiation image conversion panel with laser light, read the stimulated emission,
In an operation of detecting a spot on which a radiolabeled target molecule is fixed, noise increases. Moreover,
In many cases, target molecules are attached to spots on the porous sheet simply by adhering to the porous sheet without going through hybridization, and remain between spots on the porous sheet. In such a case, noise also occurs.

Accordingly, an object of the present invention is to provide a detection method using a porous sheet that enables highly accurate and highly efficient detection of a substance to be detected that exhibits a biochemically specific reaction.

[0010]

The inventor of the present invention has conducted research on a porous sheet for detecting a bio-related substance using autoradiography. As a result, the porous sheet is partitioned by a partition wall that prevents transmission of radiation. By forming a large number of open regions and forming a composite material sheet in which the open regions are filled with a porous structure, when a probe molecule solution is spotted on the composite material sheet, the probe molecules are converted into a porous structure. It is possible to fix only to the body region, as a result, diffusion of the probe molecules at the time of spotting can be prevented, and therefore, noise of the obtained radiographic image can be reduced, and by adopting such a configuration, ,
It has been found that a porous material-filled region corresponding to a probe molecule spot formed by spotting in the conventional method can be provided at a high density.

The present inventor has also determined the detection of a radioactively labeled sample substance bound to a composite material sheet by a biochemically specific reaction by using a stimulable phosphor layer formed by separating a stimulable phosphor layer from each other in a sea-island manner. By performing autoradiography using a phosphor sheet, a radiographic image showing the existence position and / or amount of the sample substance bonded to the porous structure region of the composite material sheet by a biochemical specific reaction can be enhanced. It has been found that it is possible to obtain a sample substance which is biochemically and specifically bound to a probe molecule, with high sensitivity and high accuracy, thereby easily and reliably detecting the sample substance.

Therefore, the present invention provides a composite material sheet comprising a partition for subdividing a sheet plane in a two-dimensional direction, and a porous structure disposed in each of a plurality of regions partitioned by the partition. A detection sheet in which a probe molecule capable of binding to a sample substance and a biochemical specific reaction is attached to each porous structure (also referred to as immobilization), and the detection sheet is provided at each position of the porous structure. A step of preparing a stimulable phosphor sheet having a plurality of stimulable phosphor layers provided independently at corresponding positions; the detection sheet and the sample substance having a radioactive label are mixed in the presence of an aqueous medium. Contacting the sample substance with the probe molecules of the detection sheet in a biochemically specific manner; contacting the detection sheet and the stimulable phosphor sheet with each porous structure region of the detection sheet. The stimulable phosphor layers of the stackable phosphor sheet are superimposed in a position where they face each other, and are left in this superimposed state to be fixedly bonded to the porous structure region of the detection sheet. Absorbing and accumulating radiation energy emitted from the radioactive label of the sample substance in the stimulable phosphor layer of the stimulable phosphor sheet; irradiating the stimulable phosphor sheet having absorbed and accumulating the radiation energy with excitation light; Generating stimulable luminescent light from each of the stimulable phosphor layers of the stimulable phosphor sheet; condensing the stimulable luminescent light, converting the collected light into an electric signal by photoelectric conversion; and Processing the electrical signal,
A method for detecting a sample substance using a biochemical specific binding reaction.

[0013] The present invention also provides a composite material sheet comprising: a partition for subdividing a sheet plane in a two-dimensional direction; and a porous structure disposed in each of a plurality of regions partitioned by the partition. There is also a sample substance detection kit including a stimulable phosphor sheet having a plurality of stimulable phosphor layers provided independently of each other at positions corresponding to the respective positions of the porous structure of the composite material sheet.

The present invention also provides a composite material sheet comprising a partition for subdividing a sheet plane in a two-dimensional direction and a porous structure disposed in each of a plurality of regions partitioned by the partition. A detection sheet in which a probe molecule capable of binding to a biochemically specific reaction with a sample substance is attached to the porous structure, and independent from each other at positions corresponding to the respective positions of the porous structure on the detection sheet. There is also a detection kit comprising a stimulable phosphor sheet having a plurality of stimulable phosphor layers provided as described above.

[0015] The sample substance to be analyzed in the detection method of the present invention is preferably a substance derived from a living body, which is directly collected by extraction or isolation from the living body, and further subjected to chemical treatment or chemical treatment. Modified and the like, and replicates of these substances derived from living organisms obtained by using a replication technique such as the PCR method are included. Typical examples thereof include polynucleotides (DNA, RNA) and the like. Nucleic acids or fragments thereof; antigens, antibodies, tumor markers, enzymes, abzymes, hormones and other proteins.

The biochemical specific binding that can be used in the present invention includes the above-mentioned hybridization caused by complementation of the base sequence, and immunospecific binding such as an antigen-antibody reaction. And protein-specific binding based on the tertiary structure and the like.

The composite material sheet of the present invention, the detection sheet,
A preferred embodiment of the stimulable phosphor sheet is as follows. (1) The average density of the partition walls of the composite material sheet and the detection sheet is 0.6 g / cm ³ or more, particularly 1 to 20 g / c.
m ³ , and more preferably 2 to 10 g / cm ³ . (2) The average density of the porous structure of the composite material sheet and the detection sheet is in the range of 0.1 to 0.5 g / cm ³ . (3) The partition of the composite material sheet and the detection sheet is made of metal, plastic, and / or ceramic;
Especially preferably, it consists of nickel and / or a nickel alloy. (4) The porous structure of the composite material sheet and the detection sheet is made of a porous organic polymer substance, particularly preferably made of a polyamide and / or a cellulose derivative.

(5) The area of the opening of the porous structure of the composite material sheet and the detection sheet is less than 5 mm ² , more preferably less than 1 mm ² , still more preferably less than 0.1 mm ² , and 0.001 mm ^{It is 2} or more, particularly preferably 0.01 mm ² or more. (6) The number (density) of the porous structures of the composite material sheet and the detection sheet is 10 pieces / cm ² or more, more preferably 100 pieces / cm ² or more, and still more preferably 1000 pieces / cm ² or more.
cm ² or more, and 100000 pieces / cm ² or less, preferably 10,000 pieces / cm ² or less. (7) At least one of the surface on the front side and the surface on the back side of the sheet of the porous structure of the composite material sheet and the detection sheet is retreated to the sheet inner side from the surface of the partition wall in contact with the surface. (8) The detection method according to claim 1, wherein the probe molecule immobilized on the detection sheet is an oligonucleotide, a polynucleotide or a peptide nucleic acid, and the sample substance is a DNA fragment or a copy thereof. (9) The stimulable phosphor layers of the stimulable phosphor sheet are separated from each other by partition walls having an average density of 0.6 g / cm ³ or more.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION A composite material sheet and a detection sheet according to the present invention include a partition having a specific density for subdividing the sheet along a plane direction, and a specific partition disposed in an area defined by the partition. And a porous structure having a high density.

The structure of the composite material sheet according to the present invention comprising the partition walls and the porous structure region will be described with reference to the accompanying drawings.

FIG. 1 is a perspective view schematically showing an example of the composite material sheet of the present invention, and FIG. 2 is a sectional view taken on line II in FIG.
FIG. 4 is an enlarged partial sectional view along a line.

In FIG. 1 and FIG. 2, the composite material sheet 10 includes a partition 11 and a porous structure region 12 (hatched portion) surrounded by the partition 11. The porous structure region 12 is a minute through hole, and the area of the opening is generally less than 5 mm ² . The area of the opening of the porous structure region 12 is preferably less than 1 mm ² , more preferably less than 0.5 mm ² , and still more preferably 0.1 mm ^2.
mm ² . And preferably 0.001 mm ²
Above, and particularly preferably 0.01 mm ² or more.

The composite material sheet 1 in the porous structure region 12
It is desirable that at least one of the front side surface and the back side surface of No. 2 is retreated toward the inside of the sheet from the surface of the partition wall adjacent to the surface, as shown in FIG. With such a configuration, the probe molecule solution can be easily spotted on the porous structure region, the probe molecule solution once spotted flows out to the partition wall surface, and further, other porous structure can be spotted. This is because the outflow to the region can be effectively prevented.

The number of the porous structure regions 12 is generally one.
0 / cm ² or more, preferably 100 / cm ² or more, more preferably 500 / cm ² or more, still more preferably 1000 / cm ² or more, and preferably 100,000 / cm ² or less, And particularly preferably 1
It is 0000 / cm ² or less. All of these need not necessarily be provided at equal intervals as shown in FIG. 1, but may be divided into several blocks and a plurality of filling regions may be formed for each block.

In the present invention, in order to effectively shield radiation such as an electron beam from a radioactively labeled sample, it is effective that the partition walls 11 have an average density of 0.6 g / cm ³ or more. . The average density of the partition walls 11 is preferably in the range of 1 to 20 g / cm ³ , and particularly preferably 2 to 20 g / cm ³ .
It is in the range of 10 g / cm ³ . Since the transmission distance of the electron beam is inversely proportional to the density, the radioactive substance is ³² P, ³³ P, ³⁵ S, ¹⁴
In the case of a general radioisotope (RI) such as C, by setting the average density of the partition walls in this range, the electron beam from the RI of the sample in each region is shielded by the partition walls, and the electron beam It is possible to prevent a decrease in resolution of a radiographic image due to transmission and scattering of light.

The partition wall material having such an average density and which can be used for the composite material sheet of the present invention includes, for example, metals such as nickel and nickel alloys, and the like.
Examples include plastic materials such as polyamide resin, aramid resin, polyethylene terephthalate resin, and polyolefin resin (eg, polyethylene resin and polypropylene resin), and ceramic materials such as alumina, zirconia, magnesia, and quartz.

In the present invention, a nucleic acid or a fragment thereof,
In order to effectively immobilize a probe molecule such as a synthetic oligonucleotide or a peptide nucleic acid, the porous structure filled in the region 12 has an average density of 0.5 g / cm.
³ or less, and 0.1 g / c
It is preferably at least m ³ .

The porous structure generally has a porosity (volume ratio).
Is in the range of 10 to 90%, and the average pore diameter of the fine pores constituting the void is in the range of 0.1 to 50 μm.

Materials that can form a porous structure having such properties include cellulose derivatives such as cellulose acetate and nitrocellulose; 6-nylon, 6,6-
Polyamides (nylons) such as nylon; fluorine-based polymers such as polytetrafluoroethylene and polyvinylidene fluoride; and organic polymer materials composed of polyvinyl chloride, polycarbonate, polysulfone and polyethersulfone; and inorganic materials such as ceramics be able to. If desired, these materials can be used in combination.

In the present invention, the arrangement of the porous structure regions 12 and the shape of the openings are not limited to the lattice-like arrangement and the circular openings shown in FIG. The arrangement and shape of the body region 12 can be appropriately changed.

FIG. 3 is a top view showing an example of a variation in the arrangement of the porous structure regions. In FIG. 3, the porous structure regions 12 are arranged so as to be shifted from each other in adjacent rows.

The composite material sheet 10 of FIG. 4 and the composite material sheet 10 of FIG. 5 are top views each showing an example of a variation in the shape of the opening of the porous structure region.
In FIG. 4, the opening of the porous structure region 12 is square. In FIG. 6, the opening of the porous structure region 12 is triangular.

In addition, the porous structure regions may be randomly arranged, and the openings may be elliptical or polygonal such as hexagonal. Furthermore, it is also possible to provide an elongated rectangular porous structure region in a stripe shape and partition it only in one-dimensional direction by partition walls.

The composite material sheet of the present invention can be produced, for example, as follows. First, a substrate having a desired partition shape is manufactured using the above-described partition material.
For example, when the partition wall material is a metal, a metal can be electrodeposited on a mold by electroforming to produce a substrate having a large number of holes. When the partition wall material is plastic, a plastic sheet is prepared by applying a coating solution in which a plastic raw material is dissolved and then drying the coating, and then this is subjected to lithography such as dry etching, a LIGA process, or a laser processing method using an excimer laser or the like. By performing the etching process utilizing the above, a large number of holes can be formed in a desired shape. When the partition wall material is ceramic, after forming the slurry of the ceramic raw material into a sheet-shaped molded body under pressure, a large number of holes can be formed in the molded body by etching such as laser processing.

Next, the above-mentioned material for forming a porous structure is dissolved or dispersed in an appropriate solvent to prepare a solution or dispersion of the material, which is poured into each hole of the substrate, and
By drying, micropores can be formed in the shape of the dried film to make it porous. In the case of nylon, since the polymer shrinks due to water, a non-aqueous solution is poured into the holes and dried to form a film, and then the sheet is immersed in an aqueous solution to form micropores in the film. In the case of ceramic, a dispersion is prepared using a ceramic raw material in which desired micropores have been formed in advance. Alternatively, the composite material sheet
It can also be manufactured by using a method of press-fitting a separately prepared porous sheet into a partition material sheet having a large number of micropores. In this way, it is possible to manufacture a composite material sheet in which the porous structure is filled in each hole of a substrate having fine holes as shown in FIGS.

As the probe molecules to be fixed to the composite material sheet of the present invention, for example, various polynucleotides and oligonucleotides which can be conventionally used as probe molecules for a macro array can be used. For example, cDNA (complementary D synthesized using mRNA as a template)
NA), a part of cDNA, a polynucleotide prepared by amplification by a PCR method such as EST ("PCR product"), and a synthesized oligonucleotide. Further, artificial nucleic acids in which phosphodiester bonds of DNA are converted into peptide bonds, that is, peptide nucleic acids (PNA), or derivatives thereof may be used.
Furthermore, proteins that can generate biochemical specific binding, such as antigens and antibodies, can also be used.

Examples of specific combinations of probe molecules and target molecules include DNA (DNA or a fragment thereof, or oligo DNA) and DNA, DNA and RN
A, PNA and DNA or RNA, PNA and PNA, antigen and antibody, avidin and biotin, and the like.

The method for detecting a biopolymer substance, a fragment thereof, or a duplicate thereof using the composite material sheet of the present invention as a detection sheet is usually performed by using each of the porous structures of the composite material sheet. A sheet for detecting radioactive label in which probe molecules are spotted and immobilized is prepared, and the sheet for detecting radioactive label is attached to the radioactively labeled target molecule (sample substance) obtained by attaching a radioactive label to the compound to be detected. A method is used in which hybridization is performed on the detection sheet by contacting the sheet underneath, and the detection sheet and a stimulable phosphor sheet (radiation image conversion panel) are overlapped to perform autoradiography. Autoradiography using this stimulable phosphor sheet has advantages such as that the detection operation can be performed at room temperature and that the detection sensitivity is high.

The general structure of the stimulable phosphor sheet has already been described in various publications, and autoradiography using the stimulable phosphor sheet has been put to practical use.

The stimulable phosphor sheet is usually coated with a phosphor layer forming dispersion comprising stimulable phosphor particles and a binder on a support, dried to form a stimulable phosphor layer. It is manufactured by a method of providing a protective layer on the stimulable phosphor layer. Note that the stimulable phosphor layer can also be formed on the support by a method in which a stimulable phosphor sheet is separately and separately manufactured and attached to the support.

The stimulable phosphor sheet used in the present invention comprises a plurality of stimulable phosphors provided independently of each other at positions corresponding to the positions of the porous structures of the composite material sheet and the detection sheet. It is a stimulable phosphor sheet having a body layer. In other words, the stimulable phosphor layers are not formed as continuous layers, but are separated and independent from each other as fine point-like stimulable phosphor layers, which are generally called sea-island structures, on a support. It exists in a state where it was done.

FIGS. 6 and 7 are sectional views showing a typical structure of the stimulable phosphor sheet used in the present invention. 6 and 7, the stimulable phosphor sheet 13 includes a support 14 and a plurality of dot-shaped stimulable phosphor layers 15 formed on the surface of the support 14 independently of each other. .

The stimulable phosphor sheet used in the present invention has a high density (for example, as shown in FIG. 8) such that the plurality of dot-shaped stimulable phosphor layers 15 on the support 24 do not transmit radiation. , 0.6 g / cm ² or more) separated from each other by a partition wall 16, and furthermore, a cover sheet 17.
Is preferably provided as a protective layer.

The dotted structure (sea-island structure) of the stimulable phosphor layer in the stimulable phosphor sheet used in the present invention is not necessarily required to be provided over the entire stimulable phosphor sheet, but is partially provided in such a manner. A stimulable phosphor layer having a simple sea-island structure may be formed.

The stimulable phosphor has a wavelength of 400-9.
Irradiation with excitation light in the range of
A stimulable phosphor that emits stimulable light in the wavelength range of nm is preferred. An example of such a stimulable phosphor is disclosed in JP-A-2-193.
No. 100 and JP-A-4-310900. Preferred stimulable phosphors include alkaline earth metal halide phosphors activated by europium or cerium (eg, BaFB).
r: Eu, BaF (Br, I): Eu), and cerium-activated rare earth oxyhalide-based stimulable phosphor.

Among these, a rare earth activated alkaline earth metal fluorinated halide stimulable phosphor represented by the basic composition formula (I): M ^II FX: zLn ‥‥ (I) is particularly preferred. However, M ^II is B
a, at least one alkaline earth metal selected from the group consisting of Sr and Ca, and Ln represents Ce, Pr, S
m, Eu, Tb, Dy, Ho, Nd, Er, Tm and Y
represents at least one rare earth element selected from the group consisting of b. X represents at least one halogen selected from the group consisting of Cl, Br and I. z is 0 <z ≦
Represents a numerical value within the range of 0.2.

In the above basic composition formula (I), M ^II represents
Ba preferably accounts for more than half. Ln is particularly preferably Eu or Ce. In the basic composition formula (I), F: X = 1: 1 appears in the notation, which indicates that it has a BaFX type crystal structure, and the stoichiometry of the final composition It does not indicate the composition. In general, a state in which many F ⁺ (X ⁻ ) centers, which are vacancies of X ⁻ ions, are generated in the BaFX crystal is preferable from the viewpoint of increasing the photostimulation efficiency with respect to light of 600 to 700 nm. At this time, F is often slightly more than X.

Although omitted in the basic composition formula (I), the following additives may be added to the basic composition formula (I) as needed. bA, wN ^I , xN ^II , yN ^III where A represents a metal oxide such as Al ₂ O ₃ , SiO ₂ and ZrO ₂ . In preventing sintering between M ^II FX particles, it is preferable to use an average particle size of the primary particles has low reactivity with M ^II FX in the following ultrafine particles 0.1 [mu] m. N ^I represents at least one kind of alkali metal compound selected from the group consisting of Li, Na, K, Rb and Cs; N ^II represents an alkaline earth metal compound consisting of Mg and / or Be; ^III is Al, Ga, In,
It represents a compound of at least one trivalent metal selected from the group consisting of Tl, Sc, Y, La, Gd and Lu. As these metal compounds, it is preferable to use halides as described in JP-A-59-75200, but it is not limited thereto.

B, w, x and y are each M ^II
This is the charged addition amount when the number of moles of FX is 1, and 0
≦ b ≦ 0.5, 0 ≦ w ≦ 2, 0 ≦ x ≦ 0.3, 0 ≦ y ≦
Represents a numerical value within each range of 0.3. These figures do not represent the element ratios contained in the final composition for additives that are reduced by baking or subsequent washing. Some of the above compounds may remain as added in the final composition.
^Some react with ^II FX or are incorporated.

In addition, if necessary, the basic composition formula (I) may further include, as necessary, a Z compound described in JP-A-55-12145.
n and Cd compounds; TiO ₂ , BeO, MgO, C which are metal oxides described in JP-A-55-160078.
aO, SrO, BaO, ZnO, Y 2 O 3, La 2 O 3, I
n ₂ O ₃ , GeO ₂ , SnO ₂ , Nb ₂ O ₅ , Ta ₂ O ₅ , Th
O ₂ ; Zr and Sc compounds described in JP-A-56-116777; B compounds described in JP-A-57-23673; As described in JP-A-57-23675.
And Si compounds; tetrafluoroboric acid compounds described in JP-A-59-27980; JP-A-59-4728.
No. 9, hexafluorosilicic acid, hexafluorotitanic acid, and hexafluorozirconic acid comprising a monovalent or divalent salt thereof; V, Cr described in JP-A-59-56480; Mn, F
e, a compound of a transition metal such as Co and Ni, or the like may be added. Further, the present invention is not limited to the phosphor containing the above-described additive, and any phosphor having a composition which is basically regarded as a rare earth activated alkaline earth metal fluorinated halide-based stimulable phosphor can be used. It may be.

The rare earth activated alkaline earth metal fluoride halide stimulable phosphor represented by the above basic composition formula (I) usually has an aspect ratio in the range of 1.0 to 5.0. . The stimulable phosphor particles used in the stimulable phosphor sheet of the present invention generally have an aspect ratio in the range of 1.0 to 2.0 (preferably 1.0 to 1.5), and the median diameter of the particle size. (Dm) is in the range of 1 μm to 10 μm (preferably 2 to 7 μm) and σ / Dm is 50% or less (preferably 40% or less) when the standard deviation of the particle size distribution is σ. It is. In addition, as the particle shape, rectangular parallelepiped, regular hexahedron, regular octahedron, 1
There are tetrahedral type, intermediate polyhedral type and irregular-shaped pulverized particles, among which tetrahedral type is preferable. However, as long as the stimulable phosphor particles satisfy the above aspect ratio, particle size, and particle size distribution, the phosphor particles need not necessarily be tetrahedral.

As described above, the stimulable phosphor sheet used in the present invention is such that each of the scattered stimulable phosphor layers is made of a material that is impermeable to radiation energy (eg, metal such as stainless steel, aluminum, copper, brass, etc.). Material, aluminum oxide,
Preferably, a wall made of a ceramic material such as magnesium oxide, silicon nitride, or carbon, or a polymer material such as polyethylene terephthalate, polyethylene naphthalate, polyurethane resin, or acrylic resin) is provided, or the stimulable phosphor emits radiation energy. It is preferable that the sheet is buried separately in a dot shape in a sheet made of an impermeable material.

Further, together with the stimulable phosphor sheet of the present invention, a spacer composed of a thin film or sheet having a hole at a position corresponding to the porous structure area of the composite material sheet and the detection sheet may be used. preferable.

The stimulable phosphor sheet used in the present invention has a stimulable phosphor layer at a position corresponding to a position where the porous structure region of the composite material sheet and the detection sheet exists. Preferably, the composite material sheet and the detection sheet have a sea-island structure such that no stimulable phosphor layer is present in the partition region. Such a stimulable phosphor layer having a sea-island structure is, for example, a method for printing a stimulable phosphor layer material coating solution in a sea-island manner on a support surface by screen printing or the like, and drying the printed coating solution. Prepare a support having a concave portion formed thereon, apply a stimulable phosphor layer material coating solution on the support surface, wipe the support surface so that the coating solution remains only in the concave portion, and then apply the coating solution. It can be manufactured using a method of drying, a method of embedding a stimulable phosphor in the holes of a sheet having a large number of holes, and attaching the stimulable phosphor to a support.

On one surface of the stimulable phosphor sheet, a light reflecting layer made of a material reflecting excitation light and / or stimulated emission light may be provided. When a support is provided on the stimulable phosphor sheet, the light reflecting layer is usually provided between the support and the stimulable sheet. By providing the light reflecting layer, the rate of excitation light and the efficiency of extracting emitted light can be increased, resulting in higher sensitivity and higher image quality.

The stimulable phosphor sheet does not necessarily need to be provided with a support or a protective film. However, the stimulable phosphor sheet is used for transportation and handling of the stimulable phosphor sheet and to avoid a change in characteristics. A support and a protective film may be provided to increase durability, weather resistance, and stain resistance. Further, the support may be transparent. In this case, the support is suitable for a reading method using a double-sided condensing method in which stimulable light is extracted from both sides of the stimulable phosphor sheet.

The support is usually a sheet or film made of a flexible resin material and having a thickness of 50 μm to 1 mm. The support may be transparent, or alternatively,
A light reflecting material (eg, alumina particles, titanium dioxide particles, barium sulfate particles) for reflecting the excitation light or the stimulated emission light may be filled, or a void may be provided. Alternatively, the support may be filled with a light-absorbing material (eg, carbon black) for absorbing excitation light or stimulated emission light. Examples of resin materials that can be used for forming the support include various resin materials such as polyethylene terephthalate, polyethylene naphthalate, aramid resin, and polyimide resin. If necessary, the support may be a metal sheet, a ceramic sheet, a glass sheet, a quartz sheet, or the like.

The protective film is desirably transparent so as to hardly affect the incidence of excitation light and the emission of stimulated emission light, and it is also desirable to provide a physical impact or a chemical influence (for example, moisture) given from the outside. Therefore, it is desirable that the stimulable phosphor sheet is chemically stable and has high physical strength so that the stimulable phosphor sheet can be sufficiently protected from the contact. As the protective film, a solution prepared by dissolving a transparent organic polymer material such as a cellulose derivative, polymethyl methacrylate, or an organic solvent-soluble fluororesin in an appropriate solvent is applied onto the phosphor layer. Formed, or an organic polymer film such as polyethylene terephthalate, or a protective film forming sheet such as a transparent glass plate, separately formed and provided on the surface of the phosphor layer using a suitable adhesive, or inorganic What formed the compound on the fluorescent substance layer by vapor deposition etc. is used. Various additives such as light-scattering fine particles such as magnesium oxide, zinc oxide, titanium dioxide, and alumina, slip agents such as perfluoroolefin resin powder and silicone resin powder, and crosslinking agents such as polyisocyanate are contained in the protective film. May be dispersedly contained. The thickness of the protective film is generally in the range of about 0.1 μm to 20 μm.

A fluororesin coating layer may be further provided on the surface of the protective film in order to enhance the contamination resistance of the protective film. The fluororesin coating layer can be formed by applying a fluororesin solution prepared by dissolving (or dispersing) a fluororesin in an organic solvent on the surface of the protective film and drying.
The fluororesin may be used alone, but is usually used as a mixture of the fluororesin and a resin having a high film forming property. Also, an oligomer having a polysiloxane skeleton or an oligomer having a perfluoroalkyl group can be used in combination. The fluororesin coating layer may be filled with a particulate filler in order to reduce interference unevenness and further improve the quality of a radiographic image. The thickness of the fluororesin coating layer is usually in the range of 0.5 μm to 20 μm. In forming the fluororesin coating layer, additional components such as a crosslinking agent, a hardening agent, an anti-yellowing agent and the like can be used. In particular, the addition of a crosslinking agent is advantageous for improving the durability of the fluororesin coating layer.

Various structures are known as the structure of the stimulable phosphor sheet. For example, auxiliary layers such as an adhesive layer, a coloring layer, an antistatic layer, an excitation light reflection layer, and a stimulated emission light reflection layer are provided. Configurations are also known. The storage phosphor sheet used in the present invention may be provided with an auxiliary layer as necessary.

As described above, the stimulable phosphor sheet generally has a basic structure in which a support is provided on the lower surface and a protective film is provided on the upper surface. Further, the stimulable phosphor sheet usually comprises stimulable phosphor particles and a binder which contains and supports the particles in a dispersed state. However, as the stimulable phosphor sheet, a sheet composed of an aggregate of the stimulable phosphor without a binder formed by a vapor deposition method or a sintering method, or a gap between the aggregates of the stimulable phosphor is used. Some are infiltrated with high molecular substances.

Next, the detection method of the present invention will be described by taking as an example a detection method using a synthetic oligonucleotide as a probe molecule to be fixed and bound to a composite material sheet and using a DNA fragment as a sample substance.

In autoradiography using a stimulable phosphor sheet, for example, a detection sheet and a stimulable phosphor sheet to which a radiolabeled sample substance (target molecule) is bound by hybridization are shown in FIG.
As shown in (2), the porous structure region 12 of the detection sheet (composite material sheet) 10 and the dot-shaped stimulable phosphor layer 15 of the stimulable phosphor sheet 13 are opposed to each other and are in close contact with each other. In this manner, the layers are stacked and stored in this stacked state at, for example, 0 to 30 ° C. for a certain period of time (eg, 1 to 120 hours). With this save process,
Radiation energy emitted from the radioactive substance bonded and fixed to the detection sheet is absorbed and recorded (stored) in the stimulable phosphor sheet together with its positional information. The stimulable phosphor sheet in which a latent image is formed by absorbing and accumulating the radiation energy from the detection sheet together with the positional information by such an autoradiography operation, and then visualizing the image through a radiation image forming step ( Or data formation of digital image information) can be performed.

FIG. 10 is a schematic diagram for explaining the radiation image forming method of the present invention using the stimulable phosphor sheet subjected to the above-mentioned autoradiography operation.

First, the stimulable phosphor sheet is peeled off from the laminate (see FIG. 9) of the detection sheet and the stimulable phosphor sheet on which the radiolabeled target molecule is fixed, and this is peeled off from the radiation image reading apparatus shown in FIG. To load.

In FIG. 10, the stimulable phosphor sheet 13
Is transported in the direction of the arrow by transport means 31 and 32 comprising two sets of nip rollers. On the other hand, the excitation light 33 such as a laser beam is emitted from the protective layer side surface (the stimulable phosphor layer side surface) of the stimulable phosphor sheet 13. Excitation light 33
From the region in the stimulable phosphor layer of the stimulable phosphor sheet 13 which has been irradiated with the light, the brightness corresponding to the stored energy level (that is, having the energy distribution information of the radiation recorded as a latent image) Emitting light 34 is emitted. The stimulated emission light 34 is directly or reflected by a mirror 39 and is collected by a light collection guide 35 provided above.
The light is converted into an electric signal by a photoelectric conversion device (photomultiplier) 36 provided at the base of the condensing guide 35, amplified by an amplifier 37, and sent to a signal processing device 38.

The signal processing device 38 performs an appropriate operation, such as addition or subtraction, on the electric signal sent from the amplifier 37 in advance based on the type of the target radiation image and the characteristics of the radiation image conversion panel. Processing is performed, and the processed signal is sent out as an image signal.

The transmitted image signal is reproduced as a visible image by an image reproducing device (not shown), whereby
An image corresponding to the spatial energy distribution of the radiation for the A microarray is reconstructed. The playback device is a CRT
Or a recording device that performs optical scanning recording on a photosensitive film, or a device that temporarily stores an image signal in an image file such as an optical disk or a magnetic disk. It may be replaced.

On the other hand, the stimulable phosphor sheet 13 is further moved in the direction of the arrow by the nip rollers 31 and 32, and the area of the sheet subjected to the reading step is then replaced with a sodium lamp, a fluorescent lamp, an infrared lamp, or the like. In an erasing step using an erasing light source (not shown). This allows
The accumulated energy remaining on the panel after the reading step is released and removed, so that the latent image due to the remaining energy does not adversely affect the next radiation image recording (photographing) step. The removal of the residual energy may be performed immediately before the radiation image recording (photographing) step, or may be performed at both times before and after the radiation image recording (photographing) step.

As the stimulable phosphor sheet, on the surface on the side of the excitation light irradiation, the reflectance for the excitation light increases with an increase in the incident angle, while the reflectance for the stimulated emission light increases on the incident angle. Using a filter provided with a multi-layer filter that does not depend on it, while transferring the stimulable phosphor sheet in which the energy distribution information of radiation is stored and recorded as a latent image in the plane direction, or using an excitation light irradiation device While moving in the plane direction of the phosphor sheet, the phosphor sheet is irradiated with excitation light linearly in a direction orthogonal to the transport direction by using an LD array, an LED array, a fluorescent light guide sheet, or the like, and the fluorescent light is emitted. The stimulated emission light emitted from the latent image of the excitation light irradiation part of the body sheet is sequentially and one-dimensionally photoelectrically detected using a line sensor or the like in which a large number of solid-state photoelectric conversion elements are linearly arranged, That Ray energy distribution information can also be used a radiation image information reading method for obtaining a electrical image signal.

[0071]

EXAMPLES Example 1 (1) Production of Substrate Having Fine Openings Nickel was electrodeposited on a mold by electroforming to produce a substrate having a large number of holes formed therein. The size of the substrate is 40m
mx 60 mm, thickness 0.2 mm, total number of holes 2
The number of holes was 400, and the density of holes was 100 holes / cm ² . The opening of each hole is circular and the area is 0.07 m
m ² . The average density of the substrate (partition) is 8.8 g
/ Cm ³ .

(2) Formation of porous structure 15% by weight of nylon 6 was added to 83% by weight of formic acid and 2% by weight of formic acid.
And mixed at room temperature for 3 hours, and then mixed and dissolved at 50 ° C. for 1 hour, and then cooled to room temperature to prepare a polymer solution. This polymer solution was injected into each hole of the perforated substrate, and then dried to form a film in each hole. Next, the substrate was immersed in an aqueous formic acid solution (20% by weight of formic acid) to form a large number of micropores in the film to make it porous. Thus, FIG. 1 comprising a nickel partition and a porous nylon 6 filled area.
The composite material sheet having the structure schematically shown in FIGS.

(3) Evaluation of Composite Material Sheet A single-stranded nucleic acid fragment (probe molecule) was adhered and fixed to the nylon 6 porous structure region of the obtained composite material sheet by a conventional method. This detection sheet is
A single-stranded nucleic acid fragment (target molecule) having complementarity to the probe molecule was immersed in an aqueous solution of a sample molecule to which a radioactive label was attached, and hybridization was performed. Next, the detection sheet is removed from the aqueous solution, washed with water and dried,
Next, a stimulable phosphor sheet (see FIG. 8) having a stimulable phosphor layer formed in a dot shape on a support and a cover sheet and the detection sheet are connected to the porous structure region of the detection sheet. The layers were aligned so that the (probe molecule-immobilized region) and the stimulable phosphor layer region face each other, and autoradiography was performed at room temperature (see FIG. 9). The stimulable phosphor sheet after the autoradiography operation was subjected to a radiation image reproducing process using the radiation image reproducing apparatus shown in FIG. 10, and the porous structure region of the composite material sheet (where the radiolabeled sample molecule was added to the probe molecule) A radiographic image of the region fixed and immobilized by the hybridization was obtained with high sensitivity and high accuracy.

Example 2 (1) Production of Porous Substrate A substrate having a large number of holes was produced in the same manner as in Example 1.

(2) Formation of porous structure 7.5% by weight of cellulose acetate (degree of acetylation: 60%)
54% by weight of methylene chloride, 35% by weight of methanol and 3.5% by weight of water were added and mixed at room temperature for 3 hours,
Next, after mixing and dissolving at 50 ° C. for 1 hour, the mixture was cooled to room temperature to prepare a polymer solution. This polymer solution was injected into each hole of the perforated substrate, and then dried in a windless state for 5 minutes. Next, the film was dried in a breeze adjusted to a temperature of 25 ° C. and a relative humidity of 60% for 20 minutes to form micropores in the film simultaneously with the film formation. In this way, a composite material sheet including the nickel partition walls and the porous cellulose acetate-filled region and having the structure schematically shown in FIGS. 1 and 2 was obtained.

(3) Evaluation of composite material sheet Single-stranded nucleic acid fragments (probe molecules) were applied to the cellulose acetate porous structure region of the obtained composite material sheet according to a conventional method.
Was attached by spotting, and the detection sheet was immersed in an aqueous solution of a sample molecule in which a single-stranded nucleic acid target molecule having complementarity to the probe molecule was radiolabeled, and hybridization was carried out. Next, the detection sheet is taken out of the aqueous solution, washed with water, and dried. Then, a stimulable phosphor sheet having a stimulable phosphor layer and a cover sheet formed in a dot shape on a support (see FIG. 8). And the detection sheet are aligned and laminated so that the porous structure region (probe molecule-immobilized region) and the stimulable phosphor layer region of the detection sheet face each other. An autoradiography operation was performed (see FIG. 9). When the stimulable phosphor sheet after the autoradiography operation was subjected to a radiation image reproducing process using the radiation image reproducing apparatus shown in FIG. 10, the porous region of the composite material sheet (the radioactively labeled sample molecules were high in the probe molecules). Radiation image of the area fixed by hybridization)
Obtained with high sensitivity and high precision.

[0077]

According to the present invention, a composite material sheet composed of a large number of porous structure regions partitioned by high-density partition walls of the present invention is used as a composite material sheet for analyzing a biological substance, whereby probe molecules such as nucleic acid fragments can be obtained. Can be spot-fixed only to the porous structure region, thereby preventing the diffusion of the probe when the probe molecule solution is spotted, and the diffusion or non-specificity of radiolabeled target molecules, which are likely to occur during analysis work. It is possible to reduce the noise of the radiographic image obtained by autoradiography by preventing the adhesion of the phosphor, and furthermore, the stimulable phosphor layer is scattered at the position corresponding to the porous structure region of the composite material sheet. By using in combination with the stimulable phosphor sheet configured as described above, a radiation image with further enhanced resolution can be obtained, The accuracy of detection and analysis can be remarkably improved.

Further, it is possible to increase the number of porous substance-filled regions corresponding to spots of the conventional porous sheet per unit area, and to increase the density of the detection sheet more than before. .

[Brief description of the drawings]

FIG. 1 is a schematic perspective view showing an example of a composite material sheet used in the present invention.

FIG. 2 is an enlarged partial sectional view taken along the line II in FIG.

FIG. 3 is a top view showing an example of a variation of the arrangement of the porous material filling regions.

FIG. 4 is a top view showing an example of a variation in the shape of the opening of the porous material filling region.

FIG. 5 is a top view showing an example of a variation in the shape of the opening of the porous material filling region.

FIG. 6 is a schematic perspective view showing an example of a stimulable phosphor sheet used in the present invention.

FIG. 7 is an enlarged partial sectional view taken along the line II in FIG. 6;

FIG. 8 is a partial sectional view of another example of the stimulable phosphor sheet used in the present invention.

FIG. 9 is a cross-sectional view illustrating an example of a stacked state of a detection sheet and a stimulable phosphor sheet in a detection operation of the present invention.

FIG. 10 is a conceptual diagram of an apparatus for reproducing a radiation image from a stimulable phosphor sheet after performing an autoradiography operation.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 11 Partition wall 12 Porous structure area 13 Storage phosphor sheet 14 Support of storage phosphor sheet 15 Point storage phosphor layer 16 Partition wall of storage phosphor sheet 31 Nip roller 32 Nip roller 33 Excitation light 34 Excitation light Light 35 Condensing guide 36 Photoelectric conversion device (photomultiplier) 37 Amplifier 38 Signal processing device 39 Mirror

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01T 1/00 G01T 1/29 D 1/29 G21K 4/00 NG G21K 4/00 C12N 15/00 A F term (reference) 2G083 AA03 AA09 BB03 CC03 CC10 DD11 DD16 DD17 EE02 EE03 2G088 EE27 FF05 GG10 GG25 GG30 HH08 JJ05 JJ09 JJ23 JJ25 JJ30 JJ37 KK32 KK35 LL11 LL12 LL13 4B024 AA11 CA01 CA11 CA20 HA13 4B063 QA01 QA13 QQ42 QQ52 QR32 QR56 QR84 QS34 QS36 QS39 QX02 QX04 QX07

Claims (11)

[Claims] 1. A porous structure of a composite material sheet comprising a partition for subdividing a sheet plane in a two-dimensional direction and a porous structure disposed in each of a plurality of regions partitioned by the partition. A detection sheet in which a probe molecule capable of binding to a sample substance via a biochemical specific reaction is attached to a body, and independent from each other at positions corresponding to the respective positions of the porous structure of the detection sheet. Preparing a stimulable phosphor sheet having a plurality of stimulable phosphor layers provided by contacting the detection sheet and a sample substance having a radioactive label in the presence of an aqueous medium, Biochemically binding the substance to the probe molecules of the detection sheet; combining the detection sheet and the stimulable phosphor sheet with each porous structure region of the detection sheet and each of the stimulable phosphor sheets Accumulation By overlapping in an arrangement where the phosphor layers face each other, and leaving it in this overlapped state, emission from the radioactive label of the sample substance fixed and fixed to the porous structure region of the detection sheet is performed. Absorbing the radiation energy in the stimulable phosphor layer of the stimulable phosphor sheet; irradiating the stimulable phosphor sheet having absorbed the radiation energy with excitation light to emit the stimulable fluorescent light of the stimulable phosphor sheet; Generating photostimulated light from each of the body layers; condensing the photostimulated light, converting the collected light into an electric signal by photoelectric conversion, and processing the electric signal. A method for detecting a sample substance using a biochemical specific binding reaction. 2. The composite material sheet according to claim 1, wherein said partition walls have an average density of 0.
2. The method according to claim 1, wherein the average density of the porous structure is 6 g / cm ³ or more, and the average density of the porous structure is 1.0 g / cm ³ or less (however, a relationship of average density of partition walls> average density of the porous structure is satisfied). Detection method as described. 3. The detection method according to claim 2, wherein the partition walls of the composite material sheet are formed of metal, plastic and / or ceramic. 4. The detection method according to claim 1, wherein the porous structure of the composite material sheet is a porous organic polymer substance structure. 5. The average value of the area of the opening of each section where the porous structure of the composite material sheet is arranged is 0.001 to 5 m.
^2. The detection method according to claim 1, wherein the value is in the range of m2. 6. The detection method according to claim 1, wherein the average density of the sections of the composite material sheet in which the porous structures are arranged is in the range of 10 to 10,000 / cm ² . 7. The porous structure of the composite material sheet, wherein at least one of the surface on the front side and the surface on the back side of the sheet is retreated toward the inside of the sheet from the surface of the partition wall in contact with the surface. The detection method according to claim 1. 8. The detection method according to claim 1, wherein the probe molecule immobilized on the detection sheet is an oligonucleotide, a polynucleotide, or a peptide nucleic acid, and the sample substance is a DNA fragment or a copy thereof. 9. The detection method according to claim 1, wherein each of the stimulable phosphor layers uses a stimulable phosphor sheet in which each of the stimulable phosphor layers is separated from each other by partition walls having an average density of 0.6 g / cm ³ or more. 10. A composite material sheet comprising a partition for subdividing a sheet plane in a two-dimensional direction and a porous structure disposed in each of a plurality of regions partitioned by the partition, and the composite material A sample substance detection kit comprising a stimulable phosphor sheet having a plurality of stimulable phosphor layers provided independently of each other at positions corresponding to the respective positions of the porous structure of the sheet. 11. A porous structure of a composite material sheet comprising a partition for subdividing a sheet plane in a two-dimensional direction and a porous structure disposed in each of a plurality of regions partitioned by the partition. A detection sheet obtained by attaching a probe molecule capable of binding to a sample substance and a biochemical specific reaction to a body, and provided independently of each other at positions corresponding to the respective positions of the porous structure of the detection sheet. A detection kit comprising a stimulable phosphor sheet having a plurality of stimulable phosphor layers.