JP2000249706A - New biological chip and analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily select a probe according to an analytic purpose and to improve reactive sensitivity by containing insoluble grains inserted into a plurality of recesses of an insoluble carrier provided with the recesses separated from each other on a flat surface and bound with a partner material capable of being peculiarly bound with a target material on the surface. SOLUTION: This biological chip 1 includes an insoluble carrier plate 2 and insoluble grains inserted into a plurality of recesses 21 provided on one flat surface 22 of the carrier plate 2. A plurality of recesses 21 are separately arranged in an aligned state in the vertical direction and horizontal direction on the flat surface of the carrier plate 2. A partner material peculiarly bound with a target material is carried on the surface of the insoluble grains. An organic or inorganic material insoluble in water or an organic solvent in the nucleic acid hybridization assay or immunological assay, e.g. silicone or glass, is preferably used for the insoluble carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel biological chip and an analytical method.

[0002]

2. Description of the Related Art There are many known methods for immobilizing a small amount of reagent on the surface of various insoluble support plates such as silicon, glass, and various plastics. By these methods, reagent spots having different reactivities (functional groups) separated from each other can be obtained on the support surface. For example, a reagent spot having various types of DNA fragments, antibodies, and the like, which is particularly miniaturized and immobilized in a large amount, is called a so-called array, and has recently attracted attention in the fields of medicine and genetic engineering. Technology.

[0003] For example, a DNA chip has been put to practical use as an array of many types of DNA oligonucleotide chains arranged on an insoluble support plate in the form of a large number of dots. The DNA chip is usually a chip of about 1 cm square, on which 1000 or more DNA fragments (DNA probes) are aligned and mounted. When a sample containing, for example, a fluorescently-labeled DNA fragment to be detected flows on the DNA chip, the DNA fragment to be detected having a sequence complementary to the probe on the DNA chip is bound to the probe, and only the bound portion is bound. Since the identification can be performed by the fluorescence, the sequence of the DNA fragment in the sample can be analyzed. Initially, this method was mainly applied to human genome analysis, but now, for example, drug development, agricultural product management, environmental analysis, or clinical genetic testing, especially cancer, infectious disease,
Alternatively, application to early diagnosis of genetic diseases and the like is also being studied.

[0004] Many methods for producing DNA chips have been reported. For example, there is known a method of solid-phase synthesis of an oligonucleotide on a support by applying a photoreaction and a photolithography technique. In this method, a plurality of types of oligonucleotides having a desired sequence at many predetermined positions can be formed one by one or simultaneously. A reactive hydroxyl group is generated at a predetermined position by ultraviolet rays (UV) and a photomask, and the hydroxyl group is reacted with a predetermined phosphoramidite whose 5 ′ position is protected by a photodegradable protecting group to form one hydroxyl group. Bind the nucleotides. This reaction is repeated while appropriately selecting the position where the reactive hydroxyl group is generated and the type of the phosphoramidite, and nucleotides are sequentially bonded to form oligonucleotides. Using this method, a chip in which tens of thousands of probes are bonded on one chip can be produced.

[0005] Alternatively, as another method for producing a DNA chip, an oligonucleotide or cDNA synthesized in advance is used.
Is immobilized on a support by a covalent bond or a non-covalent bond. For example, a method of electrostatically adsorbing on a slide glass coated with poly-L-lysine, or a functional group (for example, -NH ₂ , -COOH,
Or -CHO or the like) and a specific binding reagent to covalently bind an oligonucleotide or cDNA.

[0006]

However, the conventional method has various disadvantages. For example, in the method using photoreaction and photolithography technology, 1.28 cm
An array (chip) having about 100,000 oligonucleotides immobilized on the area ² can be formed.
It is difficult to obtain longer oligonucleotides than r. In the chip obtained by this method, the number of bases as a probe is insufficient, so that not only the target substance of interest but also other substances may be bound,
Therefore, accurate analysis cannot be performed, and there is a need for improvement in terms of accuracy. Further, in this method, since photolithography equipment is expensive, when a small amount of the target oligonucleotide is prepared over a wide range, for example, when only a specific DNA probe is to be immobilized on a support or a required DNA probe is used. On the other hand, in the case where the number of items is small, there is a disadvantage that the degree of freedom of usability is limited.

In the above-mentioned method of immobilizing a previously synthesized oligonucleotide or cDNA on a support, it is easy to immobilize a long-chain oligonucleotide, so that a chip having a high degree of freedom of use can be produced according to the purpose. However, a large number of DNA chains must be separately synthesized and prepared in advance. Moreover, in order to immobilize those DNA chains, after processing the surface of the support, the oligonucleotide or cDNA is bonded to the support by covalent bonding using a chemical cross-linking agent. so,
Further, it cannot be denied that the hybridization ability is impaired. On the other hand, in the method of immobilizing directly on a support by a non-covalent bond, since treatment with a crosslinking agent or the like is not performed, it is easy to specifically form a pair with a complementary nucleic acid sequence. There is a problem that the influence of the solution due to the addition of the reagent cannot be avoided, sufficient immobilization cannot be maintained, and the immobilized oligonucleotide or cDNA is peeled off from the support. That is, this method has a drawback that the sensitivity of the reaction is reduced, regardless of whether it is a covalent bond or a non-covalent bond. Furthermore, reaction spots (reagent spots) require sophisticated equipment to form a solution containing oligonucleotides or cDNAs by an ink-jet method or an automatic micropipetting apparatus. In this regard, photoreaction and photolithography techniques are required. It has the same problems as the applied method.

Therefore, an object of the present invention is to solve the above-mentioned problems in the prior art, to provide a high degree of freedom in selecting a probe, to enable easy and inexpensive selection of a probe according to the purpose of analysis, and It is an object of the present invention to provide a biological chip capable of improving the sensitivity of a reaction.

[0009]

According to the present invention, there is provided a biological chip used for analyzing a target substance in a test sample according to the present invention, comprising: (1) at least one flat surface;
An insoluble support provided with a plurality of recesses separated from each other on the flat surface; and (2) a partner substance inserted into the recess of the insoluble support and capable of specifically binding to the target substance. The problem can be solved by the biological chip, wherein the biological chip includes insoluble particles having a surface bound thereto. The present invention also relates to a method for analyzing a target substance in a test sample using the above-mentioned biological chip.

[0010] The "analytical method" in this specification includes a "detection method" for determining the presence or absence of a target substance and a "measurement method" for determining the amount of a target substance. Further, in this specification, “target substance” means a substance to be analyzed.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
One embodiment of the biological chip of the present invention is schematically shown in FIGS. FIG. 1 is a perspective view schematically showing one embodiment of the biological chip of the present invention, and FIG. 2 is a view showing a state of a concave portion and insoluble particles held in the concave portion in the biological chip shown in FIG. FIG. 4 is an enlarged partial cross-sectional view schematically showing

As shown in FIGS. 1 and 2, a biological chip 1 of the present invention comprises an insoluble support plate 2 and a plurality of recesses 21 provided on one flat surface 22 of the insoluble support 2. And the insoluble particles 3 inserted therein. Each of the plurality of recesses 21 provided in the insoluble support plate 2 is disposed on the flat surface 22 of the insoluble support 2 plate so as to be separated from each other while being aligned in the vertical and horizontal directions. I have. Further, on the surface of the insoluble particles 3, a partner substance 31 capable of specifically binding to a target substance is carried.

Materials that can be used for the insoluble support in the biological chip of the present invention include those substantially insoluble in water or organic solvents commonly used in nucleic acid hybridization assays or immunological assays. Certain organic or inorganic materials, such as silicon, glass, magnetic or non-magnetic metals, or plastics can be mentioned. In particular, it is preferable to use silicon, various plastics (for example, polycarbonate, polystyrene, or polypropylene), quartz, glass, or the like. In addition, a porous material can be used as the material, and examples thereof include paper, nylon, and cellulose or a cellulose derivative (for example, nitrocellulose). In the present invention, the shape of the insoluble support is not particularly limited as long as it has at least one flat surface, but is preferably a plate or a film.

A plurality of recesses separated from each other are formed on the surface of the insoluble support. The shape and size of the concave portion are not particularly limited, as long as the concave portion is formed so as to hold the insoluble particles described later sufficiently and stably. For example, the shape of the opening of the recess may be, for example, a circle, an ellipse, or a polygon. Further, the shape of the longitudinal section of the concave portion is, for example, U
Shape, V shape (see FIG. 2), rectangular shape, semicircular shape,
Or it can be a semi-elliptical shape or the like. When the opening of the concave portion is, for example, circular, the diameter is preferably 0.01 μm to 1 cm, more preferably 0.05 μm to 1 cm.
μm to 5 mm. In the case where the opening of the concave portion has another shape, the size may be substantially the same as that of the circular opening. The depth of the recess is preferably 0.01 μm to 1 cm, and preferably 0.05 μm to 5 m.
m.

The arrangement of the recesses in the insoluble support is not particularly limited as long as the positions of the recesses can be confirmed when detecting a signal derived from the label. It can be determined appropriately according to the purpose or the analyzer used to detect the signal derived from the label. It is preferable that the recesses are arranged in line with each other in that the positions of the recesses can be easily confirmed. Further, as shown in FIG. 1, a plurality of the concave portions are linearly arranged in a line at a predetermined interval, and further,
More preferably, a plurality of such rows are arranged in parallel with each other. By arranging the recesses in this way, it is easy to automate the process of detecting a signal derived from a marker.
The interval between the concave portions provided on the surface of the insoluble support is not particularly limited as long as it is separated to the extent that it is not affected by adjacent concave portions when detecting a signal derived from a label. The shortest distance between the edges of the adjacent concave portions is preferably 0.01 μm to 1 cm, and preferably 0.05 μm to 5 mm. Also, the number of the concave portions in the insoluble support is not particularly limited, and depends on the size of the insoluble support. For example, several to several tens of thousands (generally several tens to one to two) (Ten thousand) concave portions.

As a means for forming the concave portion on the flat surface on the insoluble support, a known technique can be appropriately used, and an example thereof is an etching method. In the etching method, for example, after a negative photoresist film is coated on a silicon base material whose surface is oxidized, a photomask having a desired shape [for example, a dot corresponding to the shape of an opening of a concave portion (black dot)] Using a photomask provided with a plurality of. Subsequently, after removing the resist in the unexposed portion by a development process, the oxide film in the portion exposed by removing the resist is removed. Next, when the resist remaining on the exposed portion in the exposure process is removed, a portion covered with the oxide film and a portion where the oxide film is removed and the silicon surface is exposed appear on the silicon substrate. Of these,
By etching the silicon portion not covered with the oxide film and finally removing the remaining oxide film, a concave portion can be formed on the silicon substrate surface. Alternatively, similarly, a template can be prepared by an etching method, and the insoluble support can be prepared using the template.

The thus prepared insoluble support having a concave portion can be directly applied to the biological chip of the present invention, and if necessary, the entire surface of the insoluble support or a part thereof can be made hydrophilic. It can be used after treatment or hydrophobic treatment. Alternatively, the metal can be used after being deposited. For example, it is preferable to form an oxide film inside the concave portion after forming the concave portion on the surface of the silicon substrate, because the hydrophilicity of the inner wall of the concave portion can be increased, and the retention of the solution can be improved. .

The insoluble particles retained in the concave portion formed on one flat surface of the insoluble support may be particles substantially insoluble in water or an organic solvent under the use conditions of the biological chip according to the present invention. As long as the material is not particularly limited. Such materials include substantially insoluble organic or inorganic materials commonly used in nucleic acid hybridization or immunological assays, such as silicon, glass, magnetic or non-magnetic metals, or Plastics and the like can be mentioned. In particular, glass or latex (for example, polystyrene) is preferable, and latex particles generally used in nucleic acid hybridization assays or immunological assays can be suitably used as the insoluble particles. The insoluble particles are generally spherical,
The particle size is not particularly limited.
01 μm to 1 cm, preferably 0.05 μm to 5 mm,
More preferably, it is 0.1 μm to 1 mm.

In the biological chip of the present invention, the insoluble particles are used by binding a partner substance capable of specifically binding to a target substance on the surface thereof. In the present invention, the combination of the “target substance” and the “partner substance” is not particularly limited as long as it shows specific binding. For example, a nucleic acid (eg, an oligonucleotide or a polynucleotide) and its complementary Combinations with specific nucleic acids, combinations of antigens and antibodies (or antibody fragments), receptors and their ligands (eg,
Combinations with hormones, cytokines, neurotransmitters, or lectins), combinations of enzymes with their ligands (eg, enzyme substrate analogs, coenzymes, regulators or inhibitors), enzyme analogs and their enzyme analogs And a combination of a lectin and a sugar. In addition, “enzyme analog” means a substance having high specific affinity for a substrate for an original enzyme but not showing catalytic activity. Further, each of the compounds in each of the above-mentioned combinations can be either a “target substance” and the other a “partner substance”. For example, in the “combination of antigen and antibody”, if the antigen is a “target substance”, the antibody can be a “partner substance”,
Conversely, when the antibody is the “target substance”, the antigen can be the “partner substance”.

For example, when the biological chip of the present invention is applied to a nucleic acid hybridization assay, the above-mentioned partner substance binds complementarily to a nucleic acid (eg, an oligonucleotide or a polynucleotide) as a target substance. Oligonucleotides or polynucleotides that can be used can be used. As used herein, "oligonucleotide" or "polynucleotide"
Include 2'-deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and peptide nucleic acid (PNA).
PNA is an artificial nucleic acid in which a phosphodiester bond of DNA is converted into a peptide bond. The chain length of the oligonucleotide or polynucleotide bound to the insoluble particles can be appropriately selected depending on the purpose of analysis,
For example, DNA, RNA, or PNA to be captured
Can be determined based on the chain length of the complementary sequence of

The synthesis of the oligonucleotide used as the partner substance can be generally performed using an automatic synthesizer. If necessary, an oligonucleotide having a terminal amino group or another group modified can also be produced using an automatic synthesizer. Alternatively, an unmodified oligonucleotide can be synthesized in advance using an automatic synthesizer, and the aforementioned unmodified oligonucleotide can be modified as necessary. Methods for modifying oligonucleotides are well known, and for example, amines and fluorescein can be added. Oligonucleotides modified in this way have a higher affinity for insoluble particles than unmodified oligonucleotides. Also,
Synthesis of a polynucleotide (for example, cDNA) used as a partner substance can be performed using a general genetic engineering technique, for example, a PCR (polymerase chain reaction) method.

When the biological chip of the present invention is applied to an immunological assay, an antigen (including a hapten) or an antibody that specifically binds to a target substance can be used as the partner substance. . In this case, the target substance is not particularly limited as long as it is a component generally contained in the test sample and can be detected immunologically. By way of example, various proteins, polysaccharides, lipids, cells, various environmental substances, and the like can be mentioned. More specifically, immunoglobulins (eg, IgG, IgM, or IgA), infection-related markers (eg, HBs antigen, HBs antibody, HIV-1 antibody, HIV-2 antibody, HTLV-1 antibody, or Treponemal antibody) , Tumor-associated antigens (eg, AFP, CRP,
Or CEA), coagulation and fibrinolysis markers (eg, plasminogen, antithrombin-III, D-dimer, TA
T or PPI), antiepileptic drugs (for example, hormones), various drugs (for example, digoxin), bacterial cells (for example, O-157 or Salmonella) or their endotoxins or extracellular toxins, microorganisms, enzymes And pesticide residues, environmental hormones and the like. As the antibody used as the partner substance, either a polyclonal antibody or a monoclonal antibody obtained by a well-known method can be used. Further, the antibody is
Protein [eg, an enzyme (eg, pepsin or papain)] treated [eg, an antibody fragment (eg, Fab, Fab ′, F (ab ′) ₂ , or F)
v)].

The insoluble particles are used by binding a partner substance that specifically binds to the target substance. As a means for bonding the insoluble particles and the partner substance, a bonding method known to those skilled in the art can be used, for example, a bonding method by non-covalent bonding (so-called physical adsorption) or a covalent bonding (so-called chemical bonding) Can be used. For example, in the case of using a non-covalent bonding method, a solution containing a partner substance and insoluble particles is allowed to undergo a sufficient time for both to bind,
After stirring and mixing, the insoluble particles are separated by, for example, centrifugation or filtration, whereby insoluble particles having a partner substance bound thereto can be obtained. The obtained partner substance-bound insoluble particles can be stored in a buffer or solvent capable of maintaining the stability of the partner substance until use.

On the other hand, in the case of bonding by a covalent bond (chemical bond), a partner substance can be bonded to insoluble particles having a functional group (for example, a carboxyl group, an amino group, or a hydroxyl group). As such an insoluble particle, an insoluble particle having a carboxyl group or a primary amino group on its surface can be used as it is. In addition, in the case of insoluble particles having no functional group on the surface thereof, the functional group can be used by adding the functional group to the surface of the insoluble particle by a known method.

As a method for chemically bonding an oligonucleotide to the insoluble particle having a functional group, for example, a maleimide method or a carbodiimide method in which the 5 'end of the oligonucleotide is covalently bonded to the functional group of the insoluble particle is known. ing. For example, in the maleimide method, first, a 5 ′ end of an oligonucleotide is reacted with a maleimide compound, and a maleimide group is introduced into the 5 ′ end of the oligonucleotide. Suitable maleimide compounds include sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate. Separately from the above reaction, an SH group is provided to the insoluble particles by reacting the insoluble particles having an amino group with succinimidyl-S-acetylthioacetate and then performing deacetylation using hydroxylamine. By reacting the SH group on the insoluble particle thus imparted with the maleimide group introduced at the 5 ′ end of the oligonucleotide prepared by introducing a maleimide group at the 5 ′ end, the insoluble particle bound with the oligonucleotide Can be prepared.

On the other hand, in order to bind an oligonucleotide to a support by the carbodiimide method, it is preferable to use a carrier to which a carboxyl group has previously been bound. First, a carbodiimide having an amino group is reacted with an insoluble particle support having a carboxyl group. Examples of the carbodiimide compound include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). EDC activated by reacting with sulfo-NHS can react with insoluble particles containing carboxyl groups on the surface. Then
When this is reacted with the 5 'end of the oligonucleotide having an amino group, insoluble particles to which the oligonucleotide is bound can be prepared.

As a method for bonding the oligonucleotide to the insoluble particles having a functional group, in addition to the maleimide method and the carbodiimide method, various bonding methods, for example, a method using a biotin-avidin system may be used. Can be. In addition, a partner substance other than the oligonucleotide, that is, a polyoligonucleotide, an antigen (including a hapten), or an antibody (including an antibody fragment) or the like may be used in the same manner as in the case of the above-described oligonucleotide, or another method. It can be bound to insoluble particles by known methods. The partner substance can be chemically bonded to the insoluble particles after bonding the linker substance, if necessary.

In the biological chip of the present invention, the same kind of partner substance can be bound to one insoluble particle, or a plurality of kinds of partner substances can be bound to one insoluble particle. Although it is possible to bind them, usually, only the same kind of partner substance is bound to one insoluble particle, and for each partner substance used,
It is preferable to produce insoluble particles to which each partner substance is bound.

In the biological chip of the present invention, the insoluble particles prepared as described above and having bound thereto a partner substance that specifically binds to the target substance can be used alone or together with an appropriate buffer. It is inserted and held in a plurality of recesses on the surface of the support. In the present specification, the "holding" is not limited to a state in which the entire insoluble particles are completely contained in the concave portion and a part of the particles does not protrude from the opening of the concave portion. Projecting from the opening of the recess.

As a method for inserting the insoluble particles into the concave portions, any means can be used. For example, when using latex particles as the insoluble particles, it is preferable to use optical tweezers, and when using glass particles as the insoluble particles, it is preferable to use a micromanipulator. Alternatively, the particles can be put into the concave portion by a gravity method.

The insoluble particles thus inserted into the concave portion can be retained in the concave portion of the insoluble support without any special treatment, preferably together with a suitable buffer. If necessary, for example, the insoluble particles can be fixed inside the recess by coating with a water-soluble polymer solution.

In the biological chip of the present invention, the number of insoluble particles retained per recess is not particularly limited. However, when used for quantitative methods,
It is preferable that the number of insoluble particles retained per recess is the same. In the biological chip of the present invention, since the size of the concave portion and the size of the insoluble particle can be appropriately adjusted, one insoluble particle can be easily inserted into one concave portion. In the case where a plurality of insoluble particles are inserted per recess, the partner substance carried on the insoluble particles may be the same single species, or may be a plurality of species. You can also
Usually, the same single species is preferred.

The biological chip of the present invention thus obtained can be used for the analysis method of the present invention. According to the analysis method of the present invention, the target substance is analyzed (for example, measured or analyzed) by appropriately selecting a partner substance to be bound to the insoluble particles according to the target substance which may be present in the test sample. Detection). The test sample to which the analysis method of the present invention can be applied is not particularly limited as long as it is a sample that may contain a target substance. For example, a biological sample such as an animal ( Especially humans) bodily fluids (eg, blood, serum, plasma, or cerebrospinal fluid) or excretions (eg,
Urine, feces, or pus), organs, tissues, or cells, or non-biological samples, such as water (eg, clean water, sewage, river water, lake water, or sea water), soil, or sludge Can be.

The analysis method according to the present invention comprises the steps of (1) bringing a test sample, which may contain a target substance, into contact with a partner substance on an insoluble support in a recess, and (2) the partner substance And detecting or measuring a complex of the target substance and the target substance. The contact between the test sample and the partner substance on the insoluble support in the recess is, for example,
The test can be performed by adding a test sample that may contain a target substance into the recess.

In the analysis method according to the present invention, a known detection means or measurement means can be used as a means for detecting or measuring the complex of the partner substance and the target substance. For example, a first aspect of the analysis method according to the present invention includes: (1) a step of introducing a label into a target substance that may be present in a test sample; Contacting the test sample with the partner substance on the insoluble support in the concave part, which is added into the concave part of the biological chip according to (3), and (3) a complex of the partner substance and the target substance Detecting or measuring a signal derived from the label included in the above.

The second method in the analysis method according to the present invention.
In the embodiment, (1) a test sample which may contain a target substance is added into a concave portion of the biological chip according to the present invention, and a first partner substance on an insoluble support in the concave portion is added to the test sample. Contacting with the test sample, (2) further adding another labeled second partner substance capable of specifically reacting with the target substance into the recess, And (3) detecting or measuring a signal derived from the label contained in a complex of the first partner substance on the insoluble support, the target substance, and the labeled second partner substance. The step of performing The first partner substance on the insoluble support and the labeled second partner substance need to bind to the target substance at different positions.

Further, the third method in the analysis method according to the present invention.
In the embodiment, (1) a test sample which may contain a target substance is added into a concave portion of the biological chip according to the present invention, and a partner substance on an insoluble support in the concave portion and the test sample are added. (2) DNA synthase and at least one type of four d-labeled
ATP, dTTP, dCTP, and dGTP are further added to the concave portion, and (3) the labeled dNTP introduced into the complex of the partner substance on the insoluble support and the target substance Detecting or measuring the derived signal. The third aspect can be applied when the target substance is a nucleic acid (eg, DNA, RNA, or peptide nucleic acid) and a nucleic acid capable of complementarily binding to the nucleic acid is used as a partner substance.

In the analysis method according to the present invention, when the target substance is a nucleic acid (eg, DNA, RNA, or peptide nucleic acid) and a nucleic acid complementary to the nucleic acid is used as a partner substance, for example, According to the first aspect of the analysis method of the present invention, the target substance can be analyzed. That is, to the concave portion of the insoluble support in the biological chip according to the present invention, a test sample previously labeled with a nucleic acid as a target substance is added, a partner substance on the insoluble support, and the test sample Is contacted under suitable time and conditions, if a target substance is present in the test sample, a complex of the partner substance and the target substance is formed on the insoluble particles. Since the target substance nucleic acid hybridizes with the partner substance nucleic acid bound to the insoluble particles, the presence or absence or the amount of the target substance can be detected or measured by a well-known method used in a nucleic acid hybridization assay. it can.

For example, if a labeled nucleotide is previously added to a nucleic acid as a target substance by nick translation before adding a test sample to the concave portion, a reaction between the partner substance and the target substance may occur. A signal reflecting the result of hybridization can be detected or measured by adding the additional reagents for directly expressing the label or for expressing a reaction specific to the label. Further, the target nucleic acid can be amplified in advance by a PCR method or the like. Further, at this time, if a substance serving as a label is added to the primer, the subsequent detection is facilitated and the detection sensitivity is increased, which is convenient.

In this case, that is, when the target substance is a nucleic acid and a nucleic acid capable of complementary binding to the nucleic acid is used as a partner substance, the method according to the third aspect of the analysis method of the present invention Can also analyze the target substance. That is, the test sample is added to the concave portion of the insoluble support in the biological chip according to the present invention,
When a partner substance on an insoluble support is brought into contact with the test sample under suitable time and conditions, if a target substance is present in the test sample, a complex of the partner substance and the target substance is formed. Are formed on the insoluble particles. In the case where a single-stranded portion that does not form a double strand is present in the complex between the partner substance and the target substance, DN
When A synthase and at least one of dATP, dTTP, dCTP, and dGTP, at least one of which is labeled, are added, DNA is synthesized using the single-stranded portion as a template, and the complex is simultaneously labeled. Is introduced. After completion of the DNA synthesis, the signal can be detected or measured by directly adding the label or by adding additional reagents for expressing a reaction specific to the label.

The nucleic acid synthetase used in the third embodiment can be appropriately selected according to the type of the template in the complex of the target substance and the partner substance. For example, when the template is DNA, for example, Klenow enzyme can be used, and when the template is RNA, for example, reverse transcriptase can be used.

Next, when the target substance is an antigen and an antibody is used as a partner substance, the target substance can be analyzed, for example, according to the second embodiment of the analysis method of the present invention. That is, the test sample is added to the concave portion of the insoluble support in the biological chip according to the present invention, and the partner substance on the insoluble support is brought into contact with the test sample for a suitable time and under conditions. When the target substance is present in the sample, a complex of the antibody as the partner substance and the antigen as the target substance is formed on the insoluble particles. Next, for the antigen captured (bound) on the insoluble particles, a labeled antibody (that is, a specific antibody that specifically binds to the antigen as a target substance, and a different antibody from the antibody bound to the insoluble particles) After adding and reacting a labeled antibody that recognizes the epitope), the signal is detected or detected by directly adding the label or by adding additional reagents for expressing a reaction specific to the label. Can be measured. In this case, that is, when the target substance is an antigen and an antibody is used as the partner substance, the first method in the analysis method of the present invention
According to the aspect, the target substance can be analyzed.

Further, when the target substance is an antibody and an antigen is used as a partner substance, an antibody labeled specifically with an antibody that specifically binds to the target substance is used as the labeled antibody. Can detect or measure a signal in the same manner as in the case where the target substance is an antigen and an antibody is used as a partner substance. In addition, the target substance is a receptor, a receptor ligand, an enzyme, an enzyme ligand, an enzyme analog, an enzyme substrate, a lectin, or a sugar that is a source of the enzyme analog, and specifically binds to them as a partner substance. When a substance to be used is used, except that a labeled antibody that specifically binds to the target substance is used as the labeled antibody, the target substance is an antigen and an antibody is used as a partner substance. Similarly, a signal can be detected or measured. Alternatively, by introducing a label into the target substance in advance before adding the test sample to the concave portion of the insoluble support, the target substance can be analyzed according to the first aspect of the analysis method of the present invention. can do.

The label in the labeled antibody or the label which can be introduced in advance into the target substance includes, for example, enzymatically active groups (for example, a combination of an enzyme and an enzyme substrate, a combination of an enzyme and a prosthetic group). Combinations, or combinations of enzymes and coenzymes), spin labels, fluorescent molecules (eg, fluorophores, fluorophores), chromophores and chromogens, luminescent molecules (eg, luminescent materials, chemiluminescent materials, and living organisms) A light-emitting substance), a phosphor molecule, a ligand capable of specifically binding (eg, biotin or avidin), an electroactive substance, a radioisotope, a colored particle (including a particle containing a colored substance and a particle composed of a colored substance) or Fluorescent particles (including particles containing a fluorescent substance and particles composed of a fluorescent substance) or colloid particles (for example, selenium colloid or gold colloid)
And the like. Detection or measurement of a signal derived from each label can be performed by a known means suitable for a signal derived from a label.

[0045]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 << Preparation of oligonucleotide chip >> In this example, an oligonucleotide chip using an oligonucleotide as a partner substance was prepared.

(1) Preparation of Insoluble Support Semiconductor heat treatment apparatus [tubular electric furnace ARF-112-1 type;
Asahi Rika Manufacturing Co., Ltd.]
cm × 3 cm) was subjected to a thermal oxidation treatment (heat source = electric heater, temperature = 1000 ° C., time = 8 hours). Then
Using a spin coater [1H-DXII; Mikasa Corporation], coat a negative photoresist OMR83 (Tokyo Ohka) [500 rpm (10 seconds) and 4000 rpm (2
[0 second]], a mask was covered, and irradiation was performed for 8 seconds (light source = mercury lamp) with a mask aligner [SUSS MJB3; Carl Sus Japan Co., Ltd.] to remove the resist in the non-exposed area. In this exposure process, one side is 5
A mask in which 200 square dots (black dots) of 0 μm are linearly arranged in a line at predetermined intervals (50 μm), and furthermore, 200 lines are arranged in parallel (total number of dots = 40000) It was used.

The obtained silicon substrate is immersed in a hydrofluoric acid solution (hydrogen fluoride: ammonium fluoride = 1: 1), and after removing the oxide film on the exposed portion by removing the resist,
The resist remaining in the exposed area was also removed. Subsequently, an insoluble support having inverted pyramid-shaped concave portions was obtained by performing an etching treatment (temperature = 80 ° C., time = 1 hour) with tetramethoxyammonium hydroxide. The length of one side of the opening (square shape) of the recess is 50 μm, the depth of the recess is 27 μm, the number of recesses per row is 200 (total of 40,000), and the distance between adjacent recesses is Was 50 μm.

Next, the obtained insoluble support was used as a starting material
As a material, instead of the photoresist OMR83,
Positive photoresist OFPR-800 (Tokyo Ohka)
Except where used, the thermal oxidation, exposure,
Photoresist removal treatment, hydrofluoric acid treatment, and residual resist removal
By repeating the removing process, the SiO _Two
An insoluble support having a layer formed thereon was obtained.

(2) Preparation of Oligonucleotide-Bound Latex Particles 0.1 ml of a suspension of carboxylated latex particles (2.5% latex, funakoshi, particle size = 10 μm) and a base sequence: 5′-tttttttttt tttttttttt cccccccc c
After mixing with 0.6 ml of a 0.1 M imidazole buffer containing an oligonucleotide having ccccccccc-3 ′ (hereinafter referred to as oligonucleotide t ₂₀ c ₂₀ ) [International Reagents Co., Ltd.], 0.1 M -Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) 0.1 ml
Was added and shaken at 25 ° C. for 20 hours. Then, 2 × S
By washing with SC solution (0.3 M-NaCl and 0.1M sodium citrate), to give the oligonucleotide t ₂₀ c ₂₀ bonded to the surface of the latex particles (oligonucleotide t ₂₀ c ₂₀ coupled latex particles) . As a control, the oligonucleotide t ₂₀
Instead of c _20, nucleotide sequence: 5'-tttttttttt tttttttttt gggggggggg g
Oligonucleotides having ggggggggg-3 'By repeating the operation, except for using (hereinafter, referred to as oligonucleotides t ₂₀ g _20), said oligonucleotide t ₂₀ g ₂₀ bonded to the surface of the latex particles (oligonucleotide t
₂₀ g ₂₀ bound latex particles).

(3) Preparation of oligonucleotide chip 40000 recesses of the insoluble support obtained in (1) above
The oligonucleotide t obtained in the above section (2)₂₀c₂₀
Bound latex particles or oligonucleotide t ₂₀g₂₀Conclusion
One of the mixed latex particles, one for each recess
By inserting them using optical tweezers,
An oligonucleotide chip according to the invention was obtained. Got
Image of scanning electron microscope of oligonucleotide chip
3 is shown. FIG. 3 shows a table of the oligonucleotide chip.
5 is a scanning electron microscope photograph replacing the drawing showing the structure of the surface.
You.

[0051]

[Embodiment 2] This embodiment "Determination of oligonucleotide in the test sample", as a test sample, the 5 'end of the oligonucleotide t ₂₀ g _20, introducing fluorescein isothiocyanate (FITC), a fluorescent substance The labeled aqueous oligonucleotide solution (4.6 μg / ml) was used.
20 μl of the above labeled oligonucleotide aqueous solution was added to 40000 concave portions of the insoluble support in the oligonucleotide chip prepared in Example 1 and reacted at 37 ° C. for 100 minutes. After the completion of the reaction, the well was washed with a 2 × SSC solution, and the amount of fluorescence in each concave portion was measured using a fluorescence measurement device. As a result, whereas it was possible to detect the fluorescence in recess inserting the oligonucleotides t ₂₀ c ₂₀ coupled latex particles, the fluorescence from the recess by inserting the oligonucleotide t ₂₀ g ₂₀ coupled latex particles are controlled Not detected. Therefore, it was confirmed that the oligonucleotide in the test sample can be detected by using the biological chip of the present invention.

[0052]

According to the biological chip of the present invention, even if it is a long-chain oligonucleotide exceeding tens of mer, it is difficult to use it by the conventional method using photolithography technology, it can be used as a probe. Can be easily handled. Also, when the number of probes is small (for example,
(When a specific probe is used), it is possible to cope easily and at low cost. Therefore, according to the biological chip of the present invention, the degree of freedom in selecting a probe is high,
Since it is possible to select a probe according to the purpose of analysis, it is possible to easily determine the target substance contained in the test sample and to improve the accuracy of the reaction.

According to the biological chip of the present invention,
It does not require the sophisticated equipment required in the conventional method of immobilizing a previously synthesized oligonucleotide or cDNA on a support. In addition, it is possible to avoid the problems such as a decrease in the reactivity of the probe due to a chemical crosslinking agent and a drop-off (peeling) of the probe due to washing or addition of a reagent in the reaction stage, which are problems in the conventional method. The sensitivity of the reaction can be improved.

[Brief description of the drawings]

FIG. 1 is a perspective view schematically showing one embodiment of the biological chip of the present invention.

FIG. 2 is an enlarged partial cross-sectional view schematically showing a state of a concave portion and insoluble particles held in the concave portion in the biological chip of the present invention shown in FIG.

FIG. 3 is a scanning electron micrograph instead of a drawing showing the surface structure of the oligonucleotide chip according to the present invention prepared in Example 1.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Biological chip; 2 ... Insoluble support plate; 21 ... Depression; 22 ... Flat surface; 3 ... Insoluble particles;

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/68 G01N 33/50 P G01N 33/50 33/566 33/566 C12N 15/00 A Reference) 2G045 AA40 BB22 BB51 CA26 CB03 CB30 DA12 DA13 DA14 FB01 FB02 FB03 FB07 GC15 HA02 4B024 AA11 AA20 BA80 CA01 CA09 CA11 HA11 HA12 HA13 HA14 HA15 4B029 AA07 AA21 BB15 BB16 BB17 Q13 Q13 Q13 Q13 Q13 Q13 Q13 Q13 Q13 Q19 QQ19 QQ42 QR32 QR55 QR56 QR82 QS34 QX02

Claims (2)

[Claims] 1. A biological chip used for analysis of a target substance in a test sample, comprising: (1) having at least one flat surface, and providing a plurality of mutually separated recesses on the flat surface. And (2) insoluble particles having a partner substance, which is inserted into the recess of the insoluble support and capable of specifically binding to the target substance, bonded to the surface thereof. The biological chip. 2. A method for analyzing a target substance in a test sample using the biological chip according to claim 1.