JP2003270244A - Biological substance detection method, detection unit and detection device used for the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a biological substance capable of easily identifying a plurality of types of biological substances, and a detection unit and a detection device used in the method. SOLUTION: One of the biological materials is fixed to a support so as to form a predetermined shape set in accordance with the type of the biological material, so that the one of the biological materials is supported by the support, and The interaction between the biological materials is performed by contacting the other biological material that causes an observable optical change in the appearance of the support with the other biological material by the interaction and detecting the presence or absence of the optical change on the support. To detect.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a biological substance for searching or detecting a biological substance, a detection unit used for the method, and a detection device. Moreover,
Whether a predetermined shape of the immobilized biomaterial can be observed on the support without using a relative scale such as a marker when detecting the result by using the support on which the biomaterial is immobilized. The present invention relates to a biological material detection method, a detection unit and a detection device for detecting and evaluating the structure, property or performance of a biological material on the absolute scale.

[0002]

2. Description of the Related Art Biological substances are a general term for substances that are synthesized or metabolized in the living body. Specifically, nucleic acids, proteins, sugar chains,
A substance composed of lipids. These biological substances are known to interact specifically with each other,
By utilizing this interaction, the search and the detection can be performed.

For example, in the case of nucleic acid, the DN of the sequence to be investigated
By immobilizing A on a support and contacting it with a sample containing one or more kinds of nucleic acid molecule species, single-stranded nucleic acids interact with each other through hydrogen bonds between complementary strands and bases. "Hybridization"), the presence or absence of the sequence to be examined can be examined. In the case of protein,
The presence or absence of the protein of interest in the subject can be examined by utilizing the interactions based on the respective three-dimensional structures.

The detection and determination of the results obtained by these methods differ depending on the method used and the shape of the support. When a filter-shaped support such as a nylon membrane is used as the support, the biological material such as the electrophoretically fractionated nucleic acid or protein is copied onto the filter-shaped support,
For example, by carrying out autoradiography using a radioactive probe, or simultaneously using a standard substance such as a molecular weight marker copied on the membrane as a reference, it is possible to determine the copied biological substance.

In the case of a container-shaped support such as the well of a microtiter plate, the color and light of the well containing the biological substance to be measured is different from that of the well to be compared and compared, for example. The target biological material can be determined based on the difference in strength.

As described above, various types of supports used for detection and retrieval of biological substances, detection methods using the same, etc. are known. It turns out that comparison and control are essential requirements in. These comparative controls are numerical values such as signal intensity and relative position information. That is, in the case of using the above-mentioned support, the numerical value of the experimental section or the position of the obtained signal is identified and judged with respect to the numerical value or position of the comparative control.

Generally, the filter-shaped support is planar, but actually has a porous structure, and in the detection of biological substances, the reagent remaining without being involved in the detection affects the detection result. It is necessary to wash and remove as much as possible so as not to give. For this reason, in the case of a filter-shaped substrate, many washing solutions and washing must be carefully performed, and there are some operations and skill required for the detection operation, and it is easy for everyone to maintain a constant level. It is hard to say that the operation is easy to obtain results.

On the other hand, the container-shaped support is generally cylindrical, and the biological substance to be detected is immobilized on the bottom surface or wall surface of the container. However, the biological substance such as polystyrene is hydrophobic or ionic. Since it is made of a material that easily binds to other biological materials, it is necessary to block it so that other biological materials that may affect the determination result in the detection operation after immobilizing the biological material are not immobilized. However, since the shape is cylindrical, it is necessary to pay attention to the removal of the solution remaining in the portion where the solution is likely to accumulate, such as the bottom edge of the container. Since this remaining solution causes variations in the determination results, when using a container-shaped support such as a microtiter plate, after removing the solution in the container, carefully perform tapping to leave the solution. It has become common to remove the resulting solution.

Further, as a method for detecting a biological substance which does not require comparison and control, a predetermined biological substance is immobilized at a certain position on a filter paper, and a sample is developed from the lower end of the filter paper by applying the principle of paper chromatography. Then, there is known a method of determining a target biomaterial depending on whether or not the biomaterial immobilized at a certain position and the developed biomaterial react with each other and coloring is recognized at the position.

Since there is no guarantee that the developed samples will not be mixed when a plurality of samples are developed, this method is generally applied to only one item. Further, in order to develop the sample, a certain amount or more of the sample that is proportional to the area of the filter paper to be developed is required, the concentration of the target biological substance in the developing solvent becomes low, and the substance interacts with the immobilized biological substance. And may not react well, or they may react but may not be detected sufficiently, and as a result, erroneous determination may result.

[0011]

When determining the result obtained by the detection or retrieval of biological material, it is necessary to avoid erroneous determination due to contaminants. When a filter-shaped or container-shaped support is used, the reaction reagent is likely to remain, so it is necessary to perform a careful washing operation in the detection operation, and skill in all the detection operations may be required. Furthermore, in the evaluation of the results, the turbidity and the presence or absence of coloration are compared with the comparison to make a judgment, but subtly the difference must be numerically compared with a measuring instrument and compared. Although it is originally a simple test, it requires a special device, and there is still room for consideration from the viewpoint of a judgment method that can be easily implemented by anyone.

Further, in the case of detecting a plurality of types of biological substances, it is necessary to introduce a label into various types of biological substances for detection which are considered to be derived from this biological substance, which requires a great deal of operation and labor. is there.

Further, in the detection method using paper chromatography, it is necessary to develop a biological substance having a sufficient concentration according to the development area, and there is room for consideration when detecting a small amount of sample.

The present invention solves the above-mentioned problems in the detection or search of biological substances derived from an analyte and makes it possible to easily identify a plurality of types of biological substances, and a method for detection used in this method. It is an object to provide a unit and a detection device.

[0015]

Means for Solving the Problems The present invention does not require comparison and control in detecting a biological substance, and searches for the biological substance by determining whether or not the type of the detected biological substance appears a shape. The present invention relates to a detection method for detecting. Further, in order to detect or search the biological substance without digitizing the obtained result, a detection unit arranged so as to show a character, a symbol, or a pattern by which the immobilized biological substance can be recognized. I will provide a. Furthermore, a detection device having a detection cell corresponding to this detection unit is provided.

That is, according to the present invention, one biomaterial is supported on a support, and the other biomaterial which brings about an observable optical change in the appearance of the support by the interaction between the biomaterials is brought into contact with the one biomaterial. In the method for detecting a biological substance that detects the presence or absence of an optical change on the support, in supporting one biological substance, one of the biological substances is formed so as to form a predetermined shape set according to the type of the biological substance. The present invention relates to a method for detecting a biological substance, which comprises immobilizing the biological substance on a support.

Further, according to the present invention, one biomaterial is brought into contact with the other biomaterial which causes an observable optical change due to the interaction between the biomaterials, and the interaction between the biomaterials is detected by the presence or absence of the optical change. The unit for detection for, including a support and a biological material supported on the surface of the support, the biological material so as to form a predetermined shape set according to the type of biological material A biological substance detection unit fixed to the support, and the detection unit, and can accommodate the other biological substance that causes an observable optical change in the appearance of the support by the interaction of the biological substances, And at least the detection unit capable of accommodating the supporting portion of one biological substance in the detection unit, and the other biological substance and the supporting portion in the detection cell. When volumes of biological material detection device supporting portion is equal to or in contact with other biological materials, it relates.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION <Detection Method of Biological Material> In the detection method of a biological material of the present invention, one biological material is supported on a support, and the appearance of the support can be observed by interaction of the biological materials. Method of detecting a presence or absence of an optical change on a support by bringing the other biomaterial that causes a physical change into contact with the one biomaterial, and in supporting one biomaterial, the type of one biomaterial One of the biological substances is immobilized on the support so as to form a predetermined shape set according to the above.

The biological substance used in the present invention is not particularly limited as long as it is a substance capable of reacting specifically with the interaction between biological substances. Examples of such biological substances include biological substances such as nucleic acids, proteins, sugar chains, and lipids, and substances having a reaction site that causes a specific reaction due to interaction between biological substances. , Extraction from living organisms, processing of extracts (eg introduction of protecting groups),
And a known method such as synthesis.

The biological substance can be supported on the support by known techniques. For example, when the biological substance is a nucleic acid or a protein, at least the predetermined shaped portion of the support is composed of a material having a property of immobilizing the nucleic acid or the protein, such as a nitrocellulose membrane, a nylon membrane, and a carbodiimide resin membrane, Examples thereof include a method of attaching and immobilizing nucleic acid or protein. The material may be appropriately selected according to the type of biological substance to be immobilized (the one biological substance).

[0021] The one biological substance is immobilized on the support so as to form a predetermined shape set according to the type of the biological substance. The predetermined shape to be used may be an existing one or a newly created one, and examples thereof include a shape selected from any character, any symbol, and any design. The predetermined shape may be formed by one biological material, for example, it may be drawn by one biological material, or one living body around the predetermined shape as shown by the white outline of the predetermined shape. The substance may be applied and fixed. When the predetermined shape is formed by arranging minute spots, it is preferable for forming various shapes and for supporting a plurality of types of biological substances on a support.

The other biological substance is not particularly limited as long as it causes an observable optical change in the appearance of the support due to the interaction between the biological substances. That is, the other biological substance may be any substance that changes the appearance of the support when bound to the other biological substance by interaction. Such optical changes include, for example, color development, decolorization, discoloration, generation or disappearance of fluorescence,
Examples of the other biological substance that causes such an optical change include labeled biological substances. The label is added to the biological substance by a known method.

The optical change is not particularly limited as long as it can be visually identified.
In order to realize a simple detection inspection, it is preferable to be able to easily visually confirm such as discoloration without using a device. Further, in the present invention, the generation and disappearance of fluorescence can be preferably used, and in such a case, the optical change can be confirmed with a simple device such as a UV lamp.

In the present invention, the other biological material is brought into contact with the one biological material supported on the support. The other biological substance may be brought into contact with the other biological substance to such an extent that it may interact with the other biological substance, and for example, a sample solution containing the other biological substance may be attached by dropping only to a predetermined shape part. Alternatively, the arbitrary shaped portion of the support may be dipped in the other sample solution containing the biological substance. It is preferable to appropriately determine the contact condition depending on the type and properties of the biological substance to be interacted with.

When the interaction between the biomaterials works, the other biomaterial binds to the one biomaterial, and the optical change having the predetermined shape is observed from the appearance of the support. Since the predetermined shape corresponds to the type of one biomaterial, one biomaterial or the other biomaterial can be searched or detected depending on the presence or absence of the predetermined shape and its type. In addition, since the result of detection or search is obtained by identifying the observed shape, compared to conventional detection methods that require relative information when determining the presence or absence of detection,
It is difficult to make an erroneous determination of the detected biological substance, and more accurate detection or search can be performed. The "search" referred to here means to identify at least a part of the constitution of the biological substance, and for example, the identification of the base sequence of nucleic acid can be mentioned.

<Biological Substance Detection Unit> The biological substance detection unit of the present invention (hereinafter, also simply referred to as “unit”) is a detection unit used in the above-mentioned method for detecting a biological substance, and comprises a support and And a biological substance supported on the surface of the support, wherein the biological substance is immobilized on the support so as to form a predetermined shape set according to the type of the biological substance. To do.

The support is not particularly limited as long as it can immobilize a biological substance such as plastic or glass. Further, the support may be made of a material capable of supporting and immobilizing the biological substance only in the portion having the predetermined shape.

The support is used to immobilize one biological substance,
Various shapes suitable for the detection operation such as contact with the other biological substance and washing after the interaction can be adopted. The shape of the support includes, for example, a cone, a cylinder, a sphere, a rectangular parallelepiped, a cube, a flat plate, a disc, and a modified body thereof. In addition, the deformable body represents a different shape as a whole including at least a part of the above-described shape, and examples thereof include a shape obtained by combining the above-described shape with another shape. A concrete example of the support is shown in FIG.
And shown in FIG.

In the detection unit of the present invention, there is no particular limitation on the number of the predetermined shapes formed on the support, but it is often necessary that a plurality of predetermined shapes be formed on the integral support. It is preferable from the viewpoint that more information can be obtained by the detection operation, and more specifically, it is 1 or more and 12 or less. This is because the size necessary for distinguishing characters, symbols, designs, etc. obtained on one unit is taken into consideration. If each character, symbol, or design is magnified using a magnifying glass or the like, it is possible to make a determination even with 12 or more types. However, in order to make the determination easily, it is preferable that the size is such that it can be visually determined.

The predetermined shape formed on the support can be formed by arranging biological substances at predetermined positions at minute points, and such a predetermined shape is generally commercially available. It can be formed by a spotting machine that can operate in the XYZ axes.

The arbitrary character refers to a character such as hiragana, katakana, kanji and alphabet which is used or recognized on the earth. Further, the predetermined shape may be a word in which a plurality of these are collected. Further, the arbitrary symbol is a symbol such as a sign used in mathematics, chemistry, physics, geography, meteorology, etc., which represents a certain meaning, and refers to all recognized on the earth. . In addition, the above-mentioned arbitrary pattern means a pattern that can be individually recognized, such as a pattern seen on clothes.

Specific examples of the predetermined shape formed by the spotting machine are shown in FIGS. One biomaterial is attached and fixed at a predetermined dot position so that the one biomaterial forms an arbitrary character, symbol, or design on the support. The arrangement of dots can be expressed on a two-dimensional grid as shown in FIG.

For example, when the symbol "+" shown in FIG. 7 is represented, the biological substance may be immobilized on the dots shown in black in the figure. In order to represent "+", in the lattice of FIG.
L, 6), (A to L, 7), (F, 1 to 12), and (G, 1 to 12) may be immobilized on the biological substance.

The support may be formed in a plurality of shapes with respect to an integral support, that is, a plurality of types of biomaterials may be immobilized. In this case, as shown in FIG. 6, they are formed independently. It is possible to immobilize two or more types of biological substances in each of the plurality of shapes that are used, and the appearance is one type, but two or more types of biological bodies are used by using a shape in which one shape includes the other shape. The substance may be immobilized on a support. In the case of utilizing a shape in which one shape includes the other shape, the predetermined shape is a shape obtained by combining two or more arbitrary shapes,
And a shape including one or more arbitrary shapes in one shape, and two or more kinds of biological substances are immobilized on a support corresponding to each of the arbitrary shapes.

For example, the symbol "+" shown in FIG. 7 corresponds to the symbol "-" shown in FIG. 8 ((A to L, 6) and (A to L, 7)).
Is included. In this case, the symbol “+” shown in FIG. 7 is formed of the biological substance A, and the symbol “−” shown in FIG. 8 is formed of the biological substance B. By doing this, when detecting biological substances,
The sign "+" is observed when the biological substance A specifically reacts with the biological substance, and the symbol "-" is observed when only the biological substance B specifically reacts with the biological substance B. It By immobilizing biomaterials in this way, it becomes possible to support the detection of multiple types of biomaterials in a support region for one shape, and it is possible to easily and accurately detect biomaterials rich in variety. Become.

The portion where the dots to be immobilized are unique to each biological substance, that is, only the biological substance which reacts only with the substance A in the above example ((F, 1 in FIG. 7
~ 5), (F, 8-12) and (G, 1-5), (G, 8
(4) to (12)), the biological substance A having an appropriate number of molecules (or concentration) from the viewpoint of signal intensity or the like may be immobilized.

The portions where the dots to be fixed are common, that is, (A to L, 6) and (A to L, 6) in the above example.
In 7), a mixture of biomaterial A and biomaterial B may be immobilized. This mixture, it is preferable to appropriately set the mixing ratio according to the signal intensity, generally, from the viewpoint of aligning the signal intensity, the number of molecules of the biological substance and the concentration of the biological substance in the specific portion, each biological substance in the mixture. It is more preferable to arrange the quantity of.

By appropriately adjusting the number of fixed molecules of the biological substance in the unique portion and the common portion, for example, adjusting the concentration of the biological substance so that the number of fixed molecules is the same, the signal intensity at the time of detection is sufficient, and It is more preferable in that a uniform optical change is detected and a uniform character or symbol or pattern is displayed.

<Biological Substance Detecting Device> The biological substance detecting device of the present invention (hereinafter, also simply referred to as “device”) can be optically confirmed when the above-mentioned unit is combined with at least one biological substance. And a detection cell (hereinafter, also simply referred to as “cell”) capable of accommodating the other biological substance in at least one unit in the unit, and supporting the other biological substance in the cell. It is characterized in that the support portion comes into contact with the other biological substance when the portion is accommodated.

The detection cell is not particularly limited as long as it can accommodate the other biological substance and the supporting portion of the unit supporting one biological substance, and the cell includes a container capable of accommodating these. . The material of the cell is not particularly limited as long as it is a material that can be stored as described above, for example, a material that is watertight when a liquid is stored in the cell, and the like.
The material may be the same as or different from that of the unit.

In the device of the present invention, a plurality of units are connected and a plurality of cells are connected correspondingly,
It becomes possible to process multiple specimens. When a plurality of units and cells are respectively connected, the individual units and cells may be bonded and connected, or may be an integrally molded product having a pre-connected structure.

As the integrally molded unit, for example, as shown in FIGS. 9 and 10, there may be mentioned an integrally molded product having a plurality of supports, and as the integrally molded cell, for example, a microtiter plate may be used. Can be mentioned. Therefore, as a more specific configuration of the device capable of processing multiple samples, the cell is a microtiter plate, and the unit has a plurality of supports corresponding to each well (preferably row or column) of the microtiter plate. An example is a configuration that is a unit.

The use of the device of the present invention will be described by taking the case of performing multi-sample treatment using nucleic acid as a sample. The nucleic acid having a different sequence is immobilized on each support of the unit, and different analyte-derived substances are contained in the cells. Contains the nucleic acid of.

In the present invention, one biological substance is immobilized on the support so as to form a predetermined shape set according to the type, so that one biological substance and the optical contact with it can be confirmed optically. Whether or not there is an interaction between the other biomaterials with the other possible biomaterial can be detected by looking at the characters or symbols or the pattern of the result obtained by one unit, and the detected shape This makes it possible to know which biological substance was detected. Therefore, as compared with the conventional detection method that requires relative information such as the intensity and position of the detected change, the detection result is less likely to be misidentified, and more accurate detection of the biological substance can be performed.

Further, in the case of supporting one of a plurality of types of biological substances on the support or in the case of supporting one of a plurality of types of biological substances on a unit having a plurality of supports, one type of biological substance of the other type is supported. By providing only the label and causing the other biological substance to act on one of the multiple types of biological substances, the interaction between the biological substances with respect to each of the multiple types of one biological substance can be detected.

Therefore, in the device of the present invention, if one type of label is provided on the other biological substance, it is possible to detect the presence or absence of the interaction with each of the plural types of one biological substance. In order to recognize each of the other biomaterials, it is not necessary to provide a plurality of types of labels on the other biomaterial according to the type of one biomaterial, and the detection operation of the biomaterial can be further simplified.

Further, in the device of the present invention, since the unit and the cell can be separated after the interaction, there is no need to wash the cell in the detection of the interaction between the biological substances, and the unit can be compared to the washing of the cell. Since it is easier to wash, it is possible to reduce the influence of the reagent used for the above-mentioned interaction on the detection result.

Further, in the device of the present invention, it is possible to detect a biological substance by bringing the other biological substance into contact with the supporting portion of one biological substance in the support.
It is also advantageous when detecting a small amount of sample, and it becomes possible to obtain many detection results from a small amount of sample.

In the device of the present invention, by preparing a plurality of cells, it becomes possible to carry out each reaction which is carried out until a detection result is obtained, in separate cells, whereby the carryover of the reagent in the previous step is carried out. It is also possible to suppress. Further, since the detection operation is completed by moving the unit between cells, the operability is also improved.

[0050]

EXAMPLES <Example 1 Preparation of DNA-immobilized unit> The DNA of SEQ ID NO: 1 was replaced with "-" on the support having the shape shown in FIG. 1 so as to have "+" shown in FIG. The DNA of SEQ ID NO: 2 was immobilized so that Each DNA was synthesized using a DNA synthesizer. A glass unit was used as the support, and the surface of the glass unit was coated with a carbodiimide resin according to the method disclosed in JP-A-11-173966.

The above-mentioned shape is formed by a set of minute points as described above. 50 pmol / DNA of SEQ ID NO: 1 was placed in the dots at the same position on the grid of each symbol.
A solution was prepared by mixing an equal amount of μL and 50 pmol / μL of the DNA of SEQ ID NO: 2 and used. In addition, each of the dots used independently has 5 of each DNA.
A 0 pmol / μL solution was prepared and used. Each solution was spotted and immobilized using a spotting machine.

Example 2 Preparation of protein-immobilized unit Anti-mouse IgG goat IgG (Vec) on the support having the shape shown in FIG.
anti-rat IgG goat IgG (Vector) was immobilized so that it was “−”. Anti-mouse IgG goat IgG is 0.5 μL / μL and anti-rat IgG goat IgG is in the same position on each grid.
A solution was prepared by mixing an equal amount of 0.5 μg / μL and used. For the dots used individually, 0.5 μg / μL solution of each IgG was prepared and used. Each solution was spotted using a spotting machine and left for 12 hours at + 4 ° C. moistening.

Then 3% BSA (bovine serum albumin)
0.1 M Tris-HCl (pH 8.0) containing
0.2 M NaCl, 0.05% Triton X-10
0 for 30 minutes at room temperature. Then 0.1M Tris
-HCl (pH 8.0), 0.2M NaCl, 0.0
It was washed with 5% Triton X-100.

<Example 3 Hybridization> The DNA shown in SEQ ID NO: 3 was synthesized by a DNA synthesizer, and 5 ′ was modified with biotin.

This biotinylated DNA was treated with 5 × SSC.
(0.45M sodium chloride, 0.045M sodium citrate), 0.05M phosphate buffer (pH 6.
5) In a solution consisting of 1 μg / mL salmon sperm DNA that has been heat-denatured and 5 × Den Hearts (0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400), 1 pmol / 100 μL It was added so as to have a concentration. 200 μL of this solution was added to the cell, the unit prepared in Example 1 was dipped, and heated at 45 ° C. for 1 hour.

Then, the unit was moved to a cell containing 2 × SSC and 0.1% SDS (sodium dodecyl sulfate), and left at room temperature for 5 minutes. Then 0.2 x SSC,
The unit was moved to a cell containing 0.1% SDS and left at 45 ° C. for 15 minutes. Then, the unit was moved to a cell containing 2 × SSC.

Next, 0.1M Tri containing 3% BSA
s-HCl (pH 8.0), 0.2 M NaCl, 0.
The unit was moved to a cell containing 05% Triton X-100 and left at room temperature for 25 minutes.

Next, the unit was transferred to a cell containing 0.1 μg / μL of streptavidin-alkaline phosphatase (Vector) and left at room temperature for 30 minutes.

Next, 0.1 M Tris-HCl (pH
8.0), 0.2M NaCl, 0.05% Trito
Move the unit to the cell containing nX-100,
It was left at room temperature for 5 minutes. Furthermore, 0.1M Tris-H
Cl (pH 8.0), 0.2M NaCl, 0.05%
The unit was moved to the cell containing Triton X-100 and left at room temperature for 5 minutes. Then 0.1M Tr
is-HCl (pH 9.5), 0.1 M NaCl,
The unit was moved to a cell containing 0.05 M magnesium chloride and left at room temperature for 1 minute.

Next, the substrate solution (10 mL of 0.1 M T
ris-HCl (pH 9.5), 0.1M NaCl,
100 mg / mL NB in 0.05M magnesium chloride
50μ of T (nitroblue tetrazolium chloride)
The above unit was moved to a cell containing 200 μL of L and 100 mg / mL BCIP (57.5-4-chloro-3-indolyl phosphoric acid) in 37.5 μL, and reacted at room temperature for 15 minutes. . Then, the unit was moved to a cell containing distilled water to stop the reaction. The results are shown in Fig. 11.

<Example 4 Antigen-antibody reaction> 1 mg of mouse IgG-alkaline phosphatase (Vector)
0.1M containing 3% BSA in 0.1 μg / mL
Tris-HCl (pH 8.0), 0.2M NaCl
It was prepared in. This was added to the cell, and the unit prepared in Example 2 was placed in the cell and reacted at room temperature for 1 hour.

Then, 0.1 M Tris-HCl (pH
8.0), 0.2M NaCl, 0.05% Trito
Move the unit to the cell containing nX-100,
It was left at room temperature for 5 minutes. Again 0.1M Tris-HC
1 (pH 8.0), 0.2M NaCl, 0.05% Tr
The unit was moved to a cell containing itonX-100 and left at room temperature for 5 minutes. After that, 0.1M Tr
is-HCl (pH 9.5), 0.1 M NaCl,
The unit was moved to a cell containing 0.05 M magnesium chloride and left at room temperature for 1 minute.

Substrate solution (10 mL of 0.1 M Tris
-HCl (pH 9.5), 0.1M NaCl, 0.05
50 mg L of 100 mg / mL NBT (nitroblue tetrazolium chloride) was added to M magnesium chloride.
0 mg / mL BCIP (5-bromo-4-chloro-3
-Indolyl phosphate) 37.5 μL added solution)
The unit was moved to a cell containing 00 μL and reacted at room temperature for 15 minutes. Then, the unit was moved to a cell containing distilled water to stop the reaction. The result is shown in FIG.
It was shown to.

[0064]

According to the present invention, one biological material is supported on a support, and the other biological material which brings about an observable optical change in the appearance of the support due to the interaction between the biological materials is used. In the method for detecting a biological substance by contacting with the substrate to detect the presence or absence of an optical change on the support, one biological substance is supported so as to form a predetermined shape set according to the type of the one biological substance. Since it is immobilized on, the biological substance can be detected by the presence or absence of the shape appearing on the support and its identification, and a plurality of types of biological substances can be easily and accurately identified.

Further, in the present invention, it is much more effective to simplify the detecting operation if the optical change observed due to the interaction between the biological substances can be visually confirmed.

Further, in the present invention, when the predetermined shape formed on the support by one of the biological substances is formed by an array of minute spots, it is more effective in easily forming the shape. .

Further, in the present invention, the presence or absence of interaction between biological substances can be detected depending on the presence or absence of the shape observed on the support, and the type of the biological substance causing the interaction can be detected depending on the type of the shape observed on the support. Since it is detected, the shape formed on the support by one biological substance can be arbitrarily selected from identifiable characters, symbols, and patterns, and the shape of the label on the other biological substance can be selected. Compared to the method of identifying the detection result, the biological substance can be detected more easily.

Further, in the present invention, when a plurality of predetermined shapes formed on the support by one biological substance are formed on the integrated support, more detection results can be obtained by one detection operation. It is even more effective, and when the number of the predetermined shapes is 1 or more and 12 or less, it is even more effective in visually confirming the optical change and its shape.

Further, in the present invention, the predetermined shape is at least one of a shape obtained by combining two or more kinds of arbitrary shapes and a shape including two or more kinds of arbitrary shapes in one shape. When two or more kinds of biomaterials are immobilized on the support corresponding to each shape, it is even more effective for easy and accurate detection of biomaterials rich in variety.

In the present invention, the support is a cone, a cylinder,
With a shape selected from a sphere, a rectangular parallelepiped, a cube, a flat plate, a disc, and these deformed bodies, it is possible to use a support having an appropriate shape according to the detection operation, so that the detection result can be easily confirmed. It is even more effective from the viewpoint of simplifying the detection operation and reducing the amount of sample used.

Further, in the present invention, when a microtiter plate is used as the detection cell and a detection unit having a plurality of supports corresponding to each well of the microtiter plate is used, in the detection operation, detection of a biological substance is conventionally performed. It is possible to utilize the equipment used for the above, facilitate the detection operation, and more effectively apply the detection device of the present invention to the detection of a large number of samples.

[0072]

[Sequence list] <110> Nisshinbo Industries, Inc. <120> Method for detecting biological material, and detection unit and detection device used therefor Su <130> P-9379 <160> 3 <210> 1 <211> 18 <212> DNA <213> Artifical Sequence <220> <223> Description of Artifical Sequence: Synthetic DNA <400> 1 atgaatagtt cgacaaag 18 <210> 2 <211> 21 <212> DNA <213> Artifical Sequence <220> <223> Description of Artifical Sequence: Synthetic DNA <400> 2 gttacccaca taccacgaat c 21 <210> 3 <211> 21 <212> DNA <213> Artifical Sequence <220> <223> Description of Artifical Sequence: Synthetic DNA <400> 3 gattcgtggt atgtgggtaa c 21

[Brief description of drawings]

FIG. 1 is a diagram showing an example of a support in a detection unit of the present invention.

FIG. 2 is a diagram showing another example of a support in the detection unit of the present invention.

FIG. 3 is a diagram showing an example of a predetermined shape formed on a support in the present invention.

FIG. 4 is a diagram showing another example of the predetermined shape formed on the support in the present invention.

FIG. 5 is a diagram showing another example of the predetermined shape formed on the support in the present invention.

FIG. 6 is a view showing another example of the predetermined shape formed on the support in the present invention.

FIG. 7 is a diagram showing an example of an arrangement of dots for forming a predetermined shape in the present invention.

FIG. 8 is a diagram showing another example of arrangement of dots for forming a predetermined shape in the present invention.

FIG. 9 is a diagram showing an example of a detection unit of the present invention having a plurality of supports.

FIG. 10 is a diagram showing another example of the detection unit of the present invention having a plurality of supports.

FIG. 11 is a diagram showing a detection result of biological substances in Example 3.

FIG. 12 is a diagram showing a detection result of biological substances in Example 4.

Claims (13)

[Claims] 1. A biological material is supported on one of the supports, and the other biological material that brings about an observable optical change in the appearance of the support by the interaction between the biological materials is brought into contact with the one biological material. In the method of detecting a biological substance for detecting the presence or absence of an optical change on the support, in the support of the one biological substance, one of the one so as to form a predetermined shape set according to the type of one biological substance. A method for detecting a biological substance, which comprises immobilizing the biological substance on a support. 2. The method for detecting a biological substance according to claim 1, wherein the optical change can be visually confirmed. 3. The predetermined shape is formed by an array of minute spots.
A method for detecting a biological substance according to the description. 4. The method for detecting a biological substance according to claim 1, wherein the predetermined shape is selected from arbitrary characters. 5. The method for detecting a biological substance according to claim 1, wherein the predetermined shape is selected from arbitrary symbols. 6. The method for detecting a biological substance according to claim 1, wherein the predetermined shape is selected from arbitrary patterns. 7. A method for detecting an interaction between biological materials by contacting one biological material with another biological material that causes an observable optical change due to the interaction between the biological materials and detecting the presence or absence of the optical change. A detection unit, which includes a support and a biological material supported on the surface of the support, wherein the biological material is formed so as to form a predetermined shape set according to the type of the biological material. A unit for detecting a biological substance, which is immobilized on a support. 8. The biological substance detection unit according to claim 7, wherein a plurality of the predetermined shapes are formed on the integrated support body. 9. The biological substance detection unit according to claim 7, wherein the number of the predetermined shapes is 1 or more and 12 or less. 10. The predetermined shape is at least one of a shape in which two or more kinds of arbitrary shapes are combined and a shape including two or more kinds of arbitrary shapes in one shape. The biological substance detection unit according to any one of claims 7 to 9, wherein two or more types of the biological substances are immobilized on the support corresponding to each of the above. 11. The biological material detection device according to claim 7, wherein the support has a shape selected from a cone, a cylinder, a sphere, a rectangular parallelepiped, a cube, a flat plate, a disc, and a modified body thereof. Unit. 12. A support and a biomaterial supported on the surface of the support, wherein one biomaterial is formed so as to form a predetermined shape according to the type of the one biomaterial. The detection unit fixed to the support, and the other biological substance that causes an observable optical change in the appearance of the support due to the interaction between the biological substances can be accommodated, and at least the one in the detection unit. And a detection cell capable of accommodating a support site for the biological substance, and when the detection site contains the other biological substance and the support site, the support site is the other biological substance. A device for detecting a biological substance, which is in contact with. 13. The biomaterial according to claim 12, wherein the detection cell is a microtiter plate, and the detection unit has a plurality of supports corresponding to each well of the microtiter plate. Device for detection.