JP3872227B2 - Novel biological chip and analytical method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel biological chip and analysis method.
[0002]
[Prior art]
Many methods for immobilizing a small amount of reagent on the surface of various insoluble support plates such as silicon, glass, or various plastics are known. 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 a large variety of DNA fragments, antibodies, etc., which is particularly miniaturized and immobilized in large quantities 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 into practical use as an array of many types of DNA oligonucleotide chains arranged on an insoluble support plate and accumulated in a number of dots. A DNA chip is usually one in which 1000 or more DNA fragments (DNA probes) are aligned and placed on a chip of about 1 cm square. For example, when a sample containing a fluorescently labeled DNA fragment to be detected is allowed to flow on this DNA chip, the DNA fragment to be detected having a sequence complementary to the probe on the DNA chip binds to the probe, and only the bound portion is bound. Since it can be distinguished by 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, pharmaceutical development, agricultural product management, environmental analysis, or clinical genetic testing, especially cancer, infectious diseases, or genetic diseases, etc. Application to early diagnosis of the disease is also being studied.
[0004]
Many methods for producing DNA chips have been reported. For example, a method for solid-phase synthesis of an oligonucleotide on a support by applying photoreaction and photolithography technology is known. In this method, a plurality of types of oligonucleotides having a desired sequence at a number of 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 a predetermined phosphoramidite protected at the 5 ′ position with a photodegradable protecting group is reacted with this hydroxyl group. Nucleotides are attached. This reaction is repeated while appropriately selecting the position where a reactive hydroxyl group is generated and the type of phosphoramidite, and nucleotides are sequentially bound to form an oligonucleotide. When this method is used, a chip in which several tens of thousands of probes are combined on one chip can be produced.
[0005]
Alternatively, as another method for producing a DNA chip, there is a method of immobilizing a pre-synthesized oligonucleotide or cDNA on a support by covalent bonding or non-covalent bonding. For example, a method of electrostatically adsorbing onto a glass slide coated with poly-L-lysine, or a functional group (for example, —NH ₂ , -COOH, or -CHO) and a specific binding reagent to covalently bind the oligonucleotide or cDNA.
[0006]
[Problems to be solved by the invention]
However, the conventional method has various drawbacks. For example, in the above-described method of applying photoreaction and photolithography technology, 1.28 cm ² An array (chip) in which about 100,000 oligonucleotides are immobilized in the same area can be formed, but it is difficult to obtain a long-chain oligonucleotide exceeding 30 mer. Since the chip obtained by this method has insufficient number of bases as a probe, it may bind not only to the target substance but also to other substances. Therefore, accurate analysis should be performed. There is a need for improvement in terms of accuracy.
In addition, in this method, since the equipment for photolithography is expensive, when preparing a small amount of the target oligonucleotide in a wide variety, for example, when it is desired to fix only a specific DNA probe on the support, or a required DNA probe On the contrary, there is a drawback in that the degree of freedom of use is limited when the number of s is sufficient.
[0007]
Further, in the above-described method of immobilizing a pre-synthesized oligonucleotide or cDNA on a support, it is easy to immobilize a long-chain oligonucleotide, so that a chip with a high degree of flexibility can be produced according to the purpose. However, it is necessary to separately synthesize and prepare a large number of DNA strands in advance.
Moreover, in order to immobilize these DNA strands, the surface of the support is processed and then covalently bonded using a chemical cross-linking agent so that the oligonucleotide or cDNA is bound to the support. Moreover, it cannot be denied that the hybridization ability is impaired. On the other hand, in the method of directly immobilizing on a support by non-covalent bond, since a treatment with a crosslinking agent or the like is not performed, it is easy to form a specific pair with a complementary nucleic acid sequence. The influence of the solution due to the addition of the reagent is unavoidable, and there is a problem that sufficient immobilization cannot be maintained and the immobilized oligonucleotide or cDNA is peeled off from the support. That is, regardless of whether the bond is a covalent bond or a non-covalent bond, this method has a drawback that the sensitivity of the reaction is lowered.
Furthermore, since the reaction spot (reagent spot) forms a solution containing an oligonucleotide or cDNA by an inkjet method or an automatic micropipetting apparatus, it requires advanced equipment. In this respect, photoreaction and photolithography technology are required. It has the same problems as the above-mentioned method to be applied.
[0008]
Accordingly, an object of the present invention is to solve the above-mentioned problems in the prior art, to have a high degree of freedom in selecting a probe, to select a probe according to the purpose of analysis easily and inexpensively, and to achieve sensitivity of the reaction. It is an object of the present invention to provide a biological chip capable of improving the efficiency.
[0009]
[Means for Solving the Problems]
The subject is a biological chip used for analysis of a target substance in a test sample according to the present invention,
(1) having at least one flat surface and having openings separated from each other on the flat surface; 0.05 μm to 5 mm And the depth is 0.05 μm to 5 mm A plate-like or film-like insoluble support provided with a plurality of recesses, and
(2) A particle size having a partner substance that is inserted into the concave portion of the insoluble support and is held non-covalently, and capable of specifically binding to the target substance, bound to the surface. 0.1 μm to 1 mm Insoluble particles that are
By means of the biological chip. Answer You can decide.
The present invention also relates to a method for analyzing a target substance in a test sample using the biological chip.
[0010]
The “analysis method” in this specification includes a “detection method” for determining the presence or absence of the target substance and a “measurement method” for determining the amount of the target substance.
In this specification, “target substance” means a substance to be analyzed.
[0011]
DETAILED DESCRIPTION OF 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 state of a recess in the biological chip shown in FIG. 1 and insoluble particles held therein. It is an enlarged partial sectional view showing typically.
[0012]
As shown in FIGS. 1 and 2, the biological chip 1 of the present invention is inserted into an insoluble support plate 2 and a plurality of recesses 21 provided on one flat surface 22 of the insoluble support 2. Insoluble particles 3.
Each of the plurality of concave portions 21 provided in the insoluble support plate 2 has the insoluble support Body rate 2 On the flat surface 22, they are arranged separately from each other in a state of being aligned in the vertical direction and the horizontal direction. The surface of the insoluble particles 3 carries a partner substance 31 that can specifically bind to the target substance.
[0013]
Materials that can be used for the insoluble support in the biological chip of the present invention include organic materials that are substantially insoluble in water or organic solvents commonly used in nucleic acid hybridization assays or immunological assays. Alternatively, an inorganic material such as silicon, glass, magnetic metal or nonmagnetic metal, or plastic can be used. In particular, it is preferable to use silicon, various plastics (for example, polycarbonate, polystyrene, polypropylene, or the like), quartz, glass, or the like.
Moreover, a porous material can also be used as said material, For example, paper, nylon, or cellulose or a cellulose derivative (for example, nitrocellulose) can be mentioned.
In the present invention, the insoluble support is not particularly limited as long as it has a shape having at least one flat surface, but is preferably plate-shaped or film-shaped.
[0014]
A plurality of recesses separated from each other are formed on the surface of the insoluble support. The shape and size of the recess are not particularly limited as long as the insoluble particles described later are formed so as to be sufficiently stably held.
For example, examples of the shape of the opening of the recess include a circle, an ellipse, and a polygon.
Furthermore, the shape of the longitudinal section of the recess may be, for example, a U shape, a V shape (see FIG. 2), a rectangular shape, a semicircular shape, a semielliptical shape, or the like.
When the opening of the recess is, for example, circular, the diameter is preferably 0.01 μm to 1 cm, more preferably 0.05 μm to 5 mm. Moreover, when the opening part of the said recessed part is another shape, it can be a magnitude | size comparable as the said circular opening part.
The depth of the recess is preferably 0.01 μm to 1 cm, preferably 0.05 μm to 5 mm.
[0015]
The arrangement of the recesses in the insoluble support is not particularly limited as long as the position of each recess can be confirmed when detecting a signal derived from a label. For example, depending on the analysis purpose Alternatively, it can be determined appropriately according to the analyzer used to detect the signal derived from the label. It is preferable that the concave portions are arranged in alignment in that the confirmation of the positions of the concave portions is easy. Moreover, as shown in FIG. 1, it is more preferable to arrange a plurality of the recesses in a line with a predetermined interval, and to arrange a plurality of such lines in parallel with each other. When the concave portion is arranged in this way, it is easy to automate the process of detecting the signal derived from the sign.
The interval between the recesses 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 the adjacent recesses when detecting a signal derived from a label, for example, The shortest distance between the edges of adjacent recesses is preferably 0.01 μm to 1 cm, preferably 0.05 μm to 5 mm.
Further, 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 tens of thousands (generally several tens to one to two). 10,000) recesses can be formed.
[0016]
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, a negative photoresist film is coated on a silicon substrate whose surface is oxidized, and then a photomask having a desired shape [for example, dots (black dots) corresponding to the shape of the opening of the recess] Using a photomask provided with a plurality of photomasks]. Subsequently, after removing the unexposed portion of the resist by development processing, the exposed oxide film is removed by removing the resist. Next, when the resist remaining in the exposed portion in the exposure process is removed, a portion covered with the oxide film and a portion where the oxide surface is removed and the silicon surface is exposed appear on the silicon substrate. Of these, the silicon portion not covered with the oxide film is etched, and finally, the remaining oxide film is removed, whereby a recess can be formed on the surface of the silicon substrate.
Alternatively, in the same manner, a template can be prepared by an etching method, and an insoluble support can be prepared using the template.
[0017]
The insoluble support having a recess prepared in this way can be applied to the biological chip of the present invention as it is, and the entire surface of the insoluble support or a part thereof is subjected to a hydrophilic treatment or a hydrophobic treatment as necessary. It can also be used after processing. Or it can also use, after vapor-depositing a metal. 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. .
[0018]
If the insoluble particles to be held in the recess formed on one flat surface of the insoluble support are particles that are substantially insoluble in water or an organic solvent under the use conditions of the biological chip according to the present invention, The material is not particularly limited. Such materials include substantially insoluble organic or inorganic materials commonly used in nucleic acid hybridization assays or immunological assays, such as silicon, glass, magnetic or non-magnetic metals, or A plastic etc. 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, and the particle size is not particularly limited, and is, for example, 0.01 μm to 1 cm, preferably 0.05 μm to 5 mm, more preferably 0.1 μm to 1 mm.
[0019]
In the biological chip of the present invention, a partner substance that can specifically bind to the target substance is bound to the surface of the insoluble particles. 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, the nucleic acid (eg, oligonucleotide or polynucleotide) and its complement Combinations of nucleic acids, antigens and antibodies (or antibody fragments), receptors and their ligands (eg, hormones, cytokines, neurotransmitters, or lectins), enzymes and their ligands (eg, enzymes) A combination of the enzyme analog and a substrate of the enzyme from which the enzyme analog is derived, or a combination of a lectin and a sugar. The “enzyme analog” means a substance that has high specific affinity with a substrate for the original enzyme but does not exhibit catalytic activity. In addition, each of the compounds in each of the above combinations can be a “target substance” and the other can be a “partner substance”. For example, in the “combination of antigen and antibody”, when the antigen becomes “target substance”, the antibody can become “partner substance”, and conversely, when the antibody becomes “target substance” The antigen can be a “partner substance”.
[0020]
For example, when the biological chip of the present invention is applied to a nucleic acid hybridization assay, the partner substance is an oligo that can be complementarily bound to a target substance, such as a nucleic acid (for example, an oligonucleotide or a polynucleotide). Nucleotides or polynucleotides can be used. As used herein, “oligonucleotide” or “polynucleotide” includes 2′-deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and peptide nucleic acid (PNA). PNA is an artificial nucleic acid obtained by converting a DNA phosphodiester bond 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, based on the chain length of the complementary sequence of DNA, RNA, or PNA to be captured. Can be determined.
[0021]
The oligonucleotide used as the partner substance can be generally synthesized using an automatic synthesizer. In addition, if necessary, an oligonucleotide having a modified terminal amino group or other group can be produced using an automatic synthesizer. Alternatively, an unmodified oligonucleotide can be synthesized in advance using an automatic synthesizer, and the 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 greater affinity for insoluble particles than unmodified oligonucleotides.
In addition, a polynucleotide (eg, cDNA) used as a partner substance can be synthesized using a normal genetic engineering technique such as a PCR (polymerase chain reaction) method.
[0022]
In addition, 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 a component that is generally contained in a test sample and that can be detected immunologically. so If there is, there is no particular limitation. For example, various proteins, polysaccharides, lipids, fungal bodies, various environmental substances, and the like can be given. More specifically, an immunoglobulin (eg, IgG, IgM, or IgA), an infection-related marker (eg, HBs antigen, HBs antibody, HIV-1 antibody, HIV-2 antibody, HTLV-1 antibody, or treponema antibody) Tumor associated antigens (eg, AFP, CRP, or CEA), coagulation fibrinolytic markers (eg, plasminogen, antithrombin-III, D-dimer, TAT, or PPI), antiepileptic drugs (eg, hormones), Examples include various drugs (for example, digoxin), bacterial cells (for example, O-157 or Salmonella) or their endotoxins or exotoxins, microorganisms, enzymes, residual agricultural chemicals, environmental hormones, and the like. As the antibody used as the partner substance, either a polyclonal antibody or a monoclonal antibody obtained by a known method can be used. Further, the antibody may be a protein [eg, enzyme (eg, pepsin or papain)] treated [eg, antibody fragment (eg, Fab, Fab ′, F (ab ′) ₂ Or Fv)].
[0023]
The insoluble particles are used by binding a partner substance that specifically binds to the target substance. As a means for binding the insoluble particles and the partner substance, a well-known binding method can be used, for example, a non-covalent bond (so-called physical adsorption) or a covalent bond (so-called chemical bond). The coupling method by can be used.
For example, when a non-covalent binding method is used, a solution containing a partner substance and insoluble particles is stirred and mixed for a time sufficient for the two to bind, and then the insoluble particles are separated, for example, by centrifugation or By separating by filtration, insoluble particles bound with the partner substance can be obtained. The resulting partner substance-bound insoluble particles can be stored in a buffer or solvent that can maintain the stability of the partner substance until use.
[0024]
On the other hand, in the case of bonding by a covalent bond (chemical bond), the 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, they can be used by adding the functional group to the surface of the insoluble particles by a known method.
[0025]
As a method for chemically bonding an oligonucleotide to an 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 a functional group of the insoluble particle is known.
For example, in the maleimide method, first, the 5 ′ end of the oligonucleotide is reacted with a maleimide compound to introduce a maleimide group at the 5 ′ end of the oligonucleotide. Suitable maleimide compounds include sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate and the like. Separately from the reaction, an insoluble particle having an amino group and succinimidyl-S-acetylthioacetate are reacted, and then deacetylation is performed using hydroxyramine to give an SH group to the insoluble particle. Insoluble particles to which oligonucleotides are bound by reacting SH groups on the insoluble particles thus imparted with maleimide groups introduced at the 5 ′ end of the oligonucleotide prepared by introducing a maleimide group at the 5 ′ end. Can be prepared.
[0026]
On the other hand, in order to bind the oligonucleotide to the support by the carbodiimide method, it is preferable to use a carrier having a carboxyl group bonded thereto in advance. 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. Next, when this is reacted with the 5 ′ end of an oligonucleotide having an amino group, insoluble particles to which the oligonucleotide is bound can be prepared.
[0027]
In addition to the maleimide method and the carbodiimide method, many binding methods, for example, a method using a biotin-avidin system can be used as a method for binding the oligonucleotide to the insoluble particles having a functional group.
In addition, partner substances other than oligonucleotides, that is, polyoligonucleotides, antigens (including haptens), antibodies (including antibody fragments), etc. may be used in the same manner as in the case of the oligonucleotides described above, It can be bound to insoluble particles by known methods. If necessary, the partner substance can be chemically bonded to the insoluble particles after binding the linker substance.
[0028]
In the biological chip of the present invention, the same kind of partner substance can be bound to one insoluble particle, or plural kinds of partner substances can be bound to one insoluble particle. However, it is usually preferable to bind only one type of partner substance to one insoluble particle and to produce insoluble particles with each partner substance for each partner substance to be used.
[0029]
In the biological chip of the present invention, the insoluble particles bound with the partner substance that specifically binds to the target substance, prepared as described above, alone or in combination with an appropriate buffer solution are used. Insert and hold in the plurality of recesses above. In the present specification, the “holding” is not limited to a state in which the entire insoluble particles are completely accommodated in the recesses, and a part of the particles does not protrude from the opening of the recess. The state which protrudes from the opening part of a recessed part is also contained.
[0030]
Arbitrary means can be used as a method of inserting the insoluble particles into the recesses. For example, when latex particles are used as insoluble particles, it is preferable to use optical tweezers, and when glass particles are used as insoluble particles, it is preferable to use a micromanipulator. Alternatively, particles can be put into the recesses by the gravitational method.
[0031]
The insoluble particles inserted into the recesses in this way can be held inside the recesses of the insoluble support, preferably together with an appropriate buffer, without any special treatment. If necessary, for example, the insoluble particles can be fixed inside the recesses by coating with a water-soluble polymer solution.
[0032]
In the biological chip of the present invention, the number of insoluble particles held per recess is not particularly limited. However, when used in a quantitative method, it is preferable that the number of insoluble particles retained per recess is the same. In the biological chip of the present invention, the size of the recess and the insoluble particles can be appropriately adjusted, so that one insoluble particle can be easily inserted into one recess.
In addition, when a plurality of insoluble particles are inserted per recess, the partner substances supported on these insoluble particles can be the same single species or can be a plurality of types. Usually, it is preferable that they are the same single species.
[0033]
The biological chip of the present invention thus obtained can be used for the analysis method according to the present invention. According to the analysis method of the present invention, the target substance is analyzed (for example, measured or measured) by appropriately selecting a partner substance that binds to the insoluble particles according to the target substance that 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 human) body fluids (eg blood, serum, plasma or cerebrospinal fluid) or excreta (eg urine, feces or pus), organs, tissues or cells, or non-biological samples, eg water (For example, water, sewage, river water, lake water, or seawater), soil, or sludge.
[0034]
The analysis method according to the present invention comprises:
(1) contacting a test sample that may contain a target substance with a partner substance on an insoluble support in a recess; and
(2) A step of detecting or measuring a complex of the partner substance and the target substance
including. The contact between the test sample and the partner substance on the insoluble support in the recess can be performed, for example, by adding a test sample that may contain a target substance into the recess.
[0035]
In the analysis method according to the present invention, known detection means or measurement means can be used as means for detecting or measuring the complex of the partner substance and the target substance.
For example, the 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;
(2) adding the obtained test sample into the recess in the biological chip according to the present invention, and bringing the partner material on the insoluble support in the recess into contact with the test sample; and
(3) A step of detecting or measuring a signal derived from the label contained in the complex of the partner substance and the target substance
including.
[0036]
The second aspect of the analysis method according to the present invention is as follows.
(1) A test sample that may contain a target substance is added into a recess in the biological chip according to the present invention, the first partner substance on the insoluble support in the recess, and the test Contacting the sample;
(2) a step of further adding another labeled second partner substance that can specifically react with the target substance into the recess; and
(3) Detect or measure 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 Process
including. The first partner substance on the insoluble support and the labeled second partner substance need to bind to the target substance at different positions.
[0037]
Furthermore, the third aspect of the analysis method according to the present invention is as follows.
(1) A test sample that may contain a target substance is added to a recess in the biological chip according to the present invention, and the partner substance on the insoluble support in the recess is combined with the test sample. Contacting,
(2) a step of further adding a DNA synthase and four types of dATP, dTTP, dCTP, and dGTP labeled with at least one type into the recess;
(3) A step of detecting or measuring a signal derived from the labeled dNTP introduced into a complex of the partner substance on the insoluble support and the target substance
including.
The third aspect can be applied when the target substance is a nucleic acid (for example, DNA, RNA, peptide nucleic acid, etc.) and a nucleic acid that can be complementarily bound to the nucleic acid is used as the partner substance.
[0038]
In the analysis method according to the present invention, when the target substance is a nucleic acid (for example, DNA, RNA, peptide nucleic acid or the like), and a nucleic acid capable of complementary binding to the nucleic acid is used as the partner substance, The target substance can be analyzed by the first aspect of the analysis method.
That is, a test sample in which a nucleic acid that is a target substance is previously labeled is added to the recess of the insoluble support in the biological chip according to the present invention, and the partner substance on the insoluble support, the test sample, and When the 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 nucleic acid that is the target substance hybridizes with the nucleic acid that is the partner substance bound to the insoluble particles, the presence or absence or amount of the nucleic acid can be detected or measured by a well-known method used in nucleic acid hybridization assays. it can.
[0039]
For example, before adding a test sample to the recess, a labeled nucleotide is added to a nucleic acid that is a target substance by nick translation in advance, and after the reaction between the partner substance and the target substance, the label The signal reflecting the hybridization result can be detected or measured directly or by adding additional reagents for expressing a reaction specific to the label.
Alternatively, the target nucleic acid can be amplified in advance by a PCR method or the like. Further, at this time, if a substance to be a label is added to the primer, it is convenient because later detection is facilitated and sensitivity of detection is increased.
[0040]
In this case, that is, when the target substance is a nucleic acid and a nucleic acid that can be complementarily bound to the nucleic acid is used as a partner substance, the target is also obtained by the third aspect of the analysis method of the present invention. The substance can be analyzed.
That is, when a test sample is added to the recess 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 under a suitable time and condition, When the target substance is present in the test sample, a complex of the partner substance and the target substance is formed on the insoluble particles. When a single-stranded portion that does not form a double strand is present in the complex of the partner substance and the target substance, next, DNA synthase and four kinds of at least one kind labeled When dATP, dTTP, dCTP, and dGTP are added, DNA is synthesized using the single-stranded portion as a template, and at the same time, a label is introduced into the complex. After completion of the DNA synthesis, the signal can be detected or measured directly by the label or by the addition of further reagents for expressing a reaction specific to the label.
[0041]
The nucleic acid synthase used in the third aspect can be appropriately selected according to the type of 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.
[0042]
Next, when the target substance is an antigen and an antibody is used as the partner substance, for example, the target substance can be analyzed by the second aspect in the analysis method of the present invention.
That is, when a test sample is added to the recess 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 under a suitable time and condition, the test is performed. When the target substance is present in the sample, a complex of the antibody that is the partner substance and the antigen that is the target substance is formed on the insoluble particles. Next, with respect to the antigen captured (bound) on the insoluble particles, a labeled antibody (that is, the antigen that specifically binds to the antigen that is a target substance, and is different from the antibody bound to the insoluble particles). And then detecting the signal directly 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 target substance can also be analyzed by the first aspect of the analysis method of the present invention.
[0043]
Further, when the target substance is an antibody and an antigen is used as the partner substance, an antibody labeled with an antibody that specifically binds to the antibody that is the target substance is used as the target antibody except that the labeled antibody is used. The signal can be detected or measured in the same manner as when the substance is an antigen and an antibody is used as the partner substance.
The target substance is a receptor, a receptor ligand, an enzyme, an enzyme ligand, an enzyme analog, an enzyme substrate that is the source of the enzyme analog, a lectin, or a sugar, and specifically binds to them as a partner substance The target substance is an antigen and the antibody is used as a partner substance, except that an antibody that specifically binds to the target substance is used as the labeled antibody. Similarly, the signal can be detected or measured. Alternatively, by adding a label to the target substance in advance before adding the test sample to the recess of the insoluble support, the target substance is also analyzed according to the first aspect of the analysis method of the present invention. can do.
[0044]
Examples of the label in the labeled antibody or the label that can be introduced into the target substance in advance include an enzymatically active group (for example, a combination of an enzyme and an enzyme substrate, a combination of an enzyme and a prosthetic group, or Combinations of enzymes and coenzymes), spin labels, fluorescent molecules (eg, fluorescent emitters, fluorophores), chromophores and chromogens, luminescent molecules (eg, luminescent, chemiluminescent, and bioluminescent) Phosphor molecules, ligands that can specifically bind (eg, biotin or avidin), electroactive substances, radioisotopes, colored particles (including particles comprising colored substances, and particles comprising colored substances) or fluorescent particles ( And particles including a fluorescent substance and particles made of the fluorescent substance), colloidal particles (for example, selenium colloid or gold colloid), and the like.
Detection or measurement of the signal derived from each label can be carried out by a known means suitable for the signal derived from the label.
[0045]
【Example】
EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these 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 produced.
[0046]
(1) Preparation of insoluble support
Using a semiconductor heat treatment apparatus [tubular electric furnace ARF-112-1 type; Asahi Rika Seisakusho Co., Ltd.], the surface of the silicon substrate (3 cm × 3 cm) is subjected to thermal oxidation treatment (heat source = electric heater, temperature = 1000 ° C., time) = 8 hours). Next, using a spin coater [1H-DXII; Mikasa Co., Ltd.], a negative photoresist OMR83 (Tokyo Ohka) was coated [500 rpm (10 seconds) and 4000 rpm (20 seconds)], and then a mask was applied. Irradiation was performed for 8 seconds with an aligner [SUSS MJB3; Carl Zoos Japan Co., Ltd.] (light source = mercury lamp) to remove the resist in the non-exposed area. In this exposure process, 200 square dots (black dots) each having a side of 50 μm are arranged in a line in a line at a predetermined interval (50 μm), and further a mask (200 lines arranged in parallel) is arranged. The total number of dots = 40000) was used.
[0047]
The obtained silicon substrate is dipped in a hydrofluoric acid solution (hydrogen fluoride: ammonium fluoride = 1: 1), and the exposed portion of the oxide film is removed by removing the resist. Removed. Subsequently, an insoluble support having an inverted pyramid-shaped recess was obtained by etching with tetramethoxyammonium hydroxide (temperature = 80 ° C., time = 1 hour). 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 40000), and between adjacent recesses Was 50 μm.
[0048]
Next, using the obtained insoluble support as a starting material, the above-described thermal oxidation treatment and exposure treatment except that a positive photoresist OFPR-800 (Tokyo Ohka) was used instead of the photoresist OMR83. , By repeating the photosensitive resist removal process, the hydrofluoric acid process, and the residual resist removal process, only the inside of the recess is made of SiO. ₂ A layered insoluble support was obtained.
[0049]
(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),
Base sequence: 5'-tttttttttt tttttttttt cccccccccc cccccccccc-3 '
(Hereinafter referred to as oligonucleotide t) ₂₀ c ₂₀ After mixing with 0.6 ml of 0.1M imidazole buffer containing [International Reagents], 0.1M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) ) 0.1 ml was added and shaken at 25 ° C. for 20 hours. Subsequently, the oligonucleotide t is washed by washing with 2 × SSC solution (0.3 M NaCl and 0.1 M sodium citrate). ₂₀ c ₂₀ Particles bound to the surface (oligonucleotide t ₂₀ c ₂₀ Bonded latex particles) were obtained.
As a control, the oligonucleotide t ₂₀ c ₂₀ Instead of,
Base sequence: 5'-tttttttttt tttttttttt gggggggggg gggggggggg-3 '
(Hereinafter referred to as oligonucleotide t) ₂₀ g ₂₀ The oligonucleotide t is repeated by repeating the above operation except that ₂₀ g ₂₀ Particles bound to the surface (oligonucleotide t ₂₀ g ₂₀ Bonded latex particles) were obtained.
[0050]
(3) Preparation of oligonucleotide chip
The oligonucleotide t obtained in the preceding item (2) is formed in 40000 concave portions of the insoluble support obtained in the preceding item (1). ₂₀ c ₂₀ Bound latex particles or oligonucleotide t ₂₀ g ₂₀ An oligonucleotide chip according to the present invention was obtained by inserting one of the bound latex particles, one for each recess, using optical tweezers.
A scanning electron micrograph of the obtained oligonucleotide chip is shown in FIG. FIG. 3 is a scanning electron micrograph in place of a drawing showing the structure of the surface of the oligonucleotide chip.
[0051]
[Example 2]
<Measurement of oligonucleotide in test sample>
In this example, the oligonucleotide t ₂₀ g ₂₀ A labeled oligonucleotide aqueous solution (4.6 μg / ml) into which fluorescein isothiocyanate (FITC), which is a fluorescent substance, was introduced at the 5 ′ end of was used.
20 μl of the 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 completion of the reaction, it was washed with 2 × SSC solution, and the amount of fluorescence in each recess was measured using a fluorescence measuring device.
As a result, oligonucleotide t ₂₀ c ₂₀ Fluorescence could be detected in the recess where the bound latex particles were inserted, whereas the control oligonucleotide t ₂₀ g ₂₀ Fluorescence was not detected from the recess where the bound latex particles were inserted. Therefore, it was confirmed that the oligonucleotide in the test sample can be detected by using the biological chip of the present invention.
[0052]
【The invention's effect】
According to the biological chip of the present invention, even long-chain oligonucleotides exceeding several tens of mer, which are difficult to use in the conventional method using photolithography technology, can be easily handled as probes. it can. Even when the number of probes is small (for example, when a specific probe is used), it can be easily and inexpensively dealt with. Therefore, according to the biological chip of the present invention, the degree of freedom in selecting a probe is high, and it is possible to select a probe according to the purpose of analysis, so that a target substance contained in a test sample can be easily discriminated. At the same time, the accuracy of the reaction can be improved.
[0053]
Further, according to the biological chip of the present invention, it is not necessary to use advanced equipment required in the conventional method for immobilizing oligonucleotides or cDNA synthesized in advance on a support. In addition, since it is possible to avoid problems such as a decrease in the reactivity of the probe due to a chemical cross-linking agent and a dropout (peeling) of the probe due to washing in the reaction stage or addition of a reagent, which was a problem 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 a biological chip of the present invention.
2 is an enlarged partial cross-sectional view schematically showing a state of a concave portion and insoluble particles held therein in the biological chip of the present invention shown in FIG. 1; FIG.
FIG. 3 is a scanning electron micrograph showing the structure of the surface of the oligonucleotide chip according to the present invention prepared in Example 1, instead of the drawing.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Biological chip; 2 ... Insoluble support plate; 21 ... Recessed part; 22 ... Flat surface; 3 ... Insoluble particle;

Claims (2)

A biological chip used for analysis of a target substance in a test sample,
(1) A plate having at least one flat surface and provided with a plurality of recesses having an opening of 0.05 μm to 5 mm and a depth of 0.05 μm to 5 mm separated from each other on the flat surface An insoluble support in the form of a film or film, and (2) a partner substance that is inserted into the recess of the insoluble support and held non-covalently and can specifically bind to the target substance. The biological chip comprising insoluble particles having a particle size of 0.1 μm to 1 mm , which are bound to each other.   A method for analyzing a target substance in a test sample using the biological chip according to claim 1.

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