JP5504587B2 - Analysis chip - Google Patents

Description

  The present invention relates to an analysis chip including a substrate on which a selective binding substance, that is, a substance that selectively binds to a specimen is immobilized.

  The analysis chip has a substrate on which a selective binding substance such as a gene, protein, lipid, sugar, etc. is immobilized. The selective binding substance on the substrate and a specimen are reacted as a normal solution, and the reaction result is obtained. Used to analyze the presence, state, quantity, etc. As the substrate, glass, metal, or resin is generally used.

  As an aspect of an analysis chip, an analysis chip in which molecules such as DNA are arranged at high density on a substrate for the purpose of simultaneously measuring a large number of gene expression of hundreds to tens of thousands, called a microarray There is something. By using a microarray, systematic and comprehensive gene expression analysis in animal models of various diseases and cell biology phenomena can be performed. Specifically, it becomes possible to clarify the function of the gene, that is, the protein encoded by the gene, and to specify the time when the protein is expressed and the place where it acts. Analyzing gene expression fluctuations at the cell or tissue level of an organism by microarray and constructing a gene expression profile database in combination with physiological, cell biological, and biochemical event data, thereby enabling disease genes and treatment-related genes Search and treatment methods.

  Analysis chips have come to be used not only for nucleic acids such as DNA, but also for testing and analyzing proteins and saccharides. In particular, in a protein analysis chip, proteins such as antibodies, antigens, and enzyme substrates are immobilized on the substrate.

  When using the analysis chip, it is important to apply the solution containing the prepared specimen so as to spread evenly over the entire region where the selective binding substance is immobilized on the analysis chip. As a contrivance for this, there is known an analysis chip including fine particles for stirring a solution containing a sample inside the analysis chip.

  In Patent Document 1, a microparticle dispersion solution in which microparticles are added in advance to a sample DNA solution is applied to an analysis chip, covered with a cover glass, sealed with a sealant, and defined by a cover glass, an analysis chip substrate, and a sealant. An analysis chip is disclosed that forms a sealed void. This analysis chip can perform hybridization without evaporating the sample solution while stirring using the movement of the fine particles.

  Patent Document 2 discloses that an analytical chip substrate is provided with irregularities, a selective binding substance is immobilized on the upper end surface of the convex portion, and the stirring fine particles are movably stored in the concave portion to stir the reaction liquid. An analysis chip that can be used is disclosed. In this analysis chip, since the fine particles do not come into contact with the upper end surface of the convex portion, stirring with the fine particles can be performed without damaging the selective binding substance.

  However, in such an analysis chip that stirs using fine particles in this way, when applying a liquid containing a specimen, bubbles remain on the surface of the analysis chip substrate or on the surface of the fine particles, or there are bubbles in the reaction solution. May occur. There was a problem that the generated bubbles obstructed and the reaction between the selective binding substance and the sample was hindered in the portion where the bubbles stayed. In addition, since reaction unevenness occurs between the portion where the bubbles stay and the other portion, there is a problem that the detection sensitivity varies on the analysis chip or the detection sensitivity decreases.

Patent Document 3 mentions that bubbles are generated when a DNA chip is used, bubbles inhibit the uniformity of the hybridization reaction, and further mentions that a foam inhibitor is added to the hybridization reaction solution composition. However, there is no mention of the actual effect of adding a foam suppressant and the type of foam suppressant used, and no investigation has been made as to what kind of foam suppressor actually has a foam suppressive effect.
Japanese Patent No. 3557419 International Publication No. 2005/090997 Pamphlet JP 2007-209236 A

  The present invention solves the above-mentioned problems, suppresses the generation of bubbles that inhibit the selective reaction between the specimen and the immobilized selective binding substance, and reduces variations in detection sensitivity due to uneven reaction and decreases in detection sensitivity. An analysis chip to be suppressed is provided.

  As a result of intensive studies, the present inventors have found that a defoaming agent (hereinafter referred to as a defoaming agent, a defoaming agent, a foaming agent, a defoaming agent) is added to a reaction solution containing a sample that reacts with the selective binding substance of the analysis chip. It was also found that the above-mentioned problems can be solved by adding a nonionic surfactant and / or a nonionic surfactant, and the present invention has been completed.

  That is, the present invention includes a substrate having a selective binding substance immobilized on the surface thereof, and a cover member bonded to the substrate, and has a gap between the substrate and the cover member, and the fine particles are formed in the gap. Is an analytical chip movably stored, and is a reaction solution for the binding reaction between the selective binding substance and the analyte in the gap, and contains an antifoaming agent and / or a nonionic surfactant. This is an analysis chip for holding a reaction solution.

  In a preferred aspect of the analysis chip of the present invention, the antifoaming agent is a polyether antifoaming agent or a silicone antifoaming agent.

  In a preferred embodiment of the analysis chip of the present invention, the surface of the fine particles is coated with a surfactant.

  In a preferred embodiment of the analysis chip of the present invention, the surfactant is an anionic surfactant or a nonionic surfactant.

  In addition, one preferable aspect of the analysis chip of the present invention is a substrate in which the substrate has a concavo-convex portion including a concave portion and a convex portion on the surface, and a selective binding substance is immobilized on the upper end surface of the convex portion. is there.

  In a preferred embodiment of the analysis chip of the present invention, the cover member has one or more through holes communicating with the gap.

  In a preferred embodiment of the analysis chip of the present invention, the shortest distance between the selective binding substance-immobilized surface of the substrate and the cover member is less than the diameter of the fine particles.

  In a preferred embodiment of the analysis chip of the present invention, the selective binding substance is DNA, RNA, protein, peptide, sugar, sugar chain or lipid.

  Furthermore, the present invention includes a substrate having a selective binding substance immobilized on the surface thereof, and a cover member bonded to the substrate, and has a gap between the substrate and the cover member, and the fine particles are formed in the gap. Including a reaction liquid containing a defoaming agent and / or a nonionic surfactant, which is a reaction liquid for the binding reaction between the selective binding substance and the sample. It is a kit for.

  According to the analysis chip of the present invention, the remaining and generation of bubbles in the reaction solution inside the analysis chip can be suppressed. As a result, the bubbles inhibit the reaction between the selective binding substance and the sample, and variations in detection sensitivity due to uneven reaction and a decrease in detection sensitivity are suppressed, and the sample can be detected with high sensitivity.

  The analysis chip of the present invention has a gap between the substrate on which the selective binding substance is fixed and the cover member bonded to the substrate, and the fine particles can be stored or injected in the gap in a movable manner. An analysis chip that is a reaction solution for a binding reaction between the selective binding substance and an analyte and that contains a defoaming agent and / or a nonionic surfactant in the void. It is characterized by that.

  An example of an analysis chip storing the fine particles of the present invention is shown in FIG. In the example shown in FIG. 1, the surface of the substrate 1 is constituted by a concavo-convex portion including a concave portion 10 and a convex portion 11. Fine particles 2 are stored in the concave portion 10, and a selective binding substance 45 (for example, nucleic acid) is immobilized on the upper surface of the convex portion 11.

  When a solution containing a specimen is added to the analysis chip in order to react with the selective binding substance 45 (for example, nucleic acid) immobilized on the substrate 1, the microbubbles attached to the surface of the microparticles 2 are released into the liquid. As shown in FIG. 2, since the bubbles 26 are formed to cover the convex portion, the selective binding substance on the covered convex portion surface cannot react with the specimen. In the analysis chip of the present invention, the reaction solution of the selective binding substance and the sample contains an antifoaming agent and / or a nonionic surfactant, so that bubbles do not adhere to the surface of the fine particles or are difficult to adhere. Therefore, generation | occurrence | production of a bubble can be suppressed. As a result, all the selective binding substances 45 on the surface of the substrate 1 can react with the specimen, and the reliability and reproducibility of the obtained data can be increased.

  Examples of the antifoaming agent contained in the reaction solution for the binding reaction between the selective binding substance and the specimen include silicone-based, polyether-based, and alcohol-based antifoaming agents.

  Examples of silicone antifoaming agents include SI manufactured by Wako Pure Chemical Industries, Airex 901W and Foamax 810 manufactured by TEGO.

  Polyether-based antifoaming agents include polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene polyoxypropylene monoalkyl ether, and three types of defoaming agents (PE-H, PE-) manufactured by Wako Pure Chemical Industries, Ltd. M, PE-L).

  Examples of the alcohol-based antifoaming agent include octyl alcohol, glycolic acid, cyclohexanol, higher alcohol, polyalkylene glycol, polyalkylene glycol alkyl ether, diethylene glycol laurate, and polyoxyalkylene sorbitan monooleate.

  Among these antifoaming agents, polyether-based or silicone-based antifoaming agents are preferable, and polyether-based antifoaming agents are more preferably used.

  The nonionic surfactant contained in the reaction solution for the binding reaction between the selective binding substance and the analyte is not particularly limited, but dimethyldecylphosphine oxide (APO-10), dodecyldimethylphosphine oxide (APO-12), Polyoxyethylene lauryl ether (BRIJ-35), polyoxyethylene (20) cetyl ether (BRIJ-58), polyoxyethylene (80) sorbitan monooleate (polysorbate 80, Tween 80), polyoxyethylene (60) sorbitan Monooleate (polysorbate 60, Tween 60), polyoxyethylene (20) sorbitan monolaurate (polysorbate 20, Tween 20), polyethylene glycol p- (1,1,3,3-tetramethylbutane) ) Phenyl ether (TRITON X-100), TRITON X-114, n-decanoyl-N-methyl-D-glucamine (MEGA-10), n-nonanoyl-N-methyl-D-glucamine (MEGA-9), n-octanoyl-N-methyl-D-glucamine (MEGA-8), nonylphenyl-polyethylene glycol (NP-40), polyoxyethylene polyoxypropylene glycol, ethylene glycol monostearate, sorbitan monostearate, monostearic acid Propylene glycol, polyoxyethylene sorbitan monostearate, polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68), polyoxyethylene (196) polyoxypropylene (67) glyco (Pluronic F127) and the like.

  Among these nonionic surfactants, polyoxyethylene (80) sorbitan monooleate (polysorbate 80, Tween 80), polyoxyethylene (60) sorbitan monooleate (polysorbate 60, Tween 60), polyoxyethylene ( 20) Sorbitan monolaurate (polysorbate 20, Tween 20), nonylphenyl-polyethylene glycol (NP-40), polyethylene glycol p- (1,1,3,3-tetramethylbutyl) phenyl ether (TRITON X-100), TRITON X-114, polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68), polyoxyethylene (196) polyoxypropylene (67) group Recall (Pluronic F127) is preferred, polyoxyethylene (80) sorbitan monooleate (polysorbate 80, Tween 80), polyoxyethylene (60) sorbitan monooleate (polysorbate 60, Tween 60), polyoxyethylene (20) Sorbitan monolaurate (polysorbate 20, Tween 20), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68), polyoxyethylene (196) polyoxypropylene (67) glycol (Pluronic F127) is more preferably used. It is done.

  The antifoaming agent and / or nonionic surfactant used in the present invention is not limited to the combination used as long as it has an effect of suppressing the generation of bubbles in the reaction solution held by the analysis chip. One or more drugs can be selected from the group consisting of an antifoaming agent and a nonionic surfactant, but preferably one or more drugs are selected from the nonionic surfactant, and the nonionic surfactant is selected. More preferably, one type is selected from the agents.

  The shape of the fine particles is not particularly limited as long as the sample solution can be stirred. In addition to the spherical shape, the shape of the fine particles may be a polygonal shape, and may be an arbitrary shape such as a microrod shape (fine rod shape) such as a cylindrical shape or a prismatic shape. be able to.

  Although the size of the fine particles is not particularly limited, it is preferable that the diameter of the fine particles is less than the shortest distance between the selective binding substance-immobilized surface of the substrate and the cover member. For example, in the case of spherical fine particles, it can be in the range of 0.1 μm to 300 μm. When the fine particles are cylindrical fine particles, the bottom diameter thereof is regarded as the diameter of the fine particles, and the bottom diameter is preferably less than the shortest distance between the selective binding substance fixing surface of the substrate and the cover member. For example, in the case of cylindrical fine particles, the length may be in the range of 50 μm to 5000 μm and the bottom diameter of 10 μm to 300 μm.

  The material of the fine particles is not particularly limited, but glass or ceramic (for example, yttrium partially stabilized zirconia), gold, platinum, stainless steel, iron, aluminum oxide (alumina), titanium oxide (titania) or other metal or metal oxide, nylon Plastics such as polystyrene can be used.

  Coating the surface of the fine particles with a surfactant is one of the preferred embodiments of the present invention because bubbles do not adhere or hardly adhere to the fine particle surfaces and generation of bubbles can be suppressed.

  As a method of coating the surface of the fine particles with a surfactant, the fine particles are immersed in a solution containing a surfactant and then taken out and dried, and the method of drying after spraying a solution containing a surfactant on the fine particle surface Well-known methods can be used, such as a method in which fine particles are brought into contact with a substance containing a surfactant solution, a method in which fine particles are brought into contact with a liquid, and a powdery surfactant is then applied. The concentration of the solution containing the surfactant used in these methods is 0.01% to 10%, more preferably 0.05% to 2%.

  Surfactants used for surface treatment of fine particles include anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants. Among these, anionic surfactants and nonionic surfactants An activator is preferably used.

  Anionic surfactants include sodium dodecyl sulfate (SDS), sodium cholate, sodium deoxycholate, sodium lauryl sarcosine, polyoxyethylene alkyl ether phosphate, polyoxyethylene alkyl phenyl ether phosphate, lauryl sulfate triethanolamine And lauroyl sarcosine sodium.

  Cationic surfactants include cetyltrimethylammonium bromide (CTAB), ethyl lanolin sulfate fatty acid aminopropylethyldimethylammonium chloride, alkyltrimethylammonium chloride, dialkyldimethylammonium chloride, distearyldimethylammonium chloride, distearyldimethylbenzylammonium chloride, And stearyltrimethylammonium chloride.

  Examples of amphoteric surfactants include 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate (CHAPSO), 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate ( CHAPS), n-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (ZWITTERGENT 3-12 Dejentent) and the like.

  Nonionic surfactants include dimethyldecylphosphine oxide (APO-10), dodecyldimethylphosphine oxide (APO-12), polyoxyethylene lauryl ether (BRIJ-35), polyoxyethylene (20) cetyl ether (BRIJ- 58), polyoxyethylene (80) sorbitan monooleate (polysorbate 80, Tween 80), polyoxyethylene (60) sorbitan monooleate (polysorbate 60, Tween 60), polyoxyethylene (20) sorbitan monolaurate ( Polysorbate 20, Tween 20), polyethylene glycol p- (1,1,3,3-tetramethylbutyl) phenyl ether (TRITON X-100), TRITON X-1 14, n-decanoyl-N-methyl-D-glucamine (MEGA-10), n-nonanoyl-N-methyl-D-glucamine (MEGA-9), n-octanoyl-N-methyl-D-glucamine (MEGA-) 8) Nonylphenyl-polyethylene glycol (NP-40), polyoxyethylene polyoxypropylene glycol, ethylene glycol monostearate, sorbitan monostearate, propylene glycol monostearate, polyoxyethylene sorbitan monostearate, polyoxyethylene (160) Polyoxypropylene (30) glycol (Pluronic F68), Polyoxyethylene (196) Polyoxypropylene (67) Glycol (Pluronic F127) and the like.

  Among these, as the anionic surfactant, sodium dodecyl sulfate (SDS) and sodium deoxycholate are particularly preferably used because of their strong surface-active effect, and as the nonionic surfactant, Pluronic F68, Pluronic F127 is particularly preferably used.

  Moreover, it is preferable that the surface of the fine particles coated with the surfactant has an appropriate roughness. That is, the center line average roughness (Ra value) on the surface of the fine particles is preferably 40 nm or more and 300 nm or less. By using fine particles having a surface roughness in this range, it is possible to coat the surface of the fine particles with a larger amount of surfactant because the surface area of the beads is larger. In the case of ceramic fine particles, considering the strength of the material, the Ra value is preferably 40 nm or more and 200 nm or less.

  It is preferable that the substrate constituting the analysis chip of the present invention has a concave and convex portion formed on the surface of the concave portion and the convex portion for fixing the selective binding substance. By adopting such a structure, a non-specifically adsorbed specimen is not detected at the time of detection, so that a noise is small and a result with a better S / N can be obtained as a result. The specific reason why noise is reduced is that when a substrate with a selective binding substance immobilized on the upper surface of the convex portion is scanned using a device called a scanner, the laser beam is focused on the upper surface of the convex portion of the concave and convex portions. This is because the laser beam is blurred in the recess, and it is difficult to detect undesired fluorescence (noise) of the specimen adsorbed nonspecifically in the recess.

  Regarding the height of the convex portions of the concave and convex portions, it is preferable that the height of the upper surface of each convex portion is substantially the same. Here, when the height is substantially the same, the selective binding substance is immobilized on the surface of the convex part, which is slightly different in height, and this is reacted with the fluorescently labeled sample, and the signal is detected when scanned with a scanner. The height at which the difference in strength of the level does not matter. Specifically, that the heights are substantially the same means that the height difference is 50 μm or less. The difference in height is more preferably 30 μm or less, and even more preferably the same height. Note that the same height as used in the present invention includes an error due to a variation that occurs in production or the like. If the difference between the height of the top surface of the highest convex portion and the height of the top surface of the lowest convex portion is larger than 50 μm, the laser beam on the top surface of the convex portion with a shifted height will be blurred and fixed to the top surface of the convex portion. The signal intensity from the sample reacted with the selective binding substance may be weak, which is not preferable.

  In the substrate constituting the analysis chip of the present invention, the region to which the selective binding substance (for example, nucleic acid) is immobilized is the surface of the substrate, as long as it is not roughened as described above. Although not particularly limited, specifically, the upper surface (upper end surface) of the convex portion of the above-described uneven portion is preferable. The selective binding substance may be immobilized in advance, or only the substrate is prepared without immobilization, and the selective binding substance corresponding to the desired specimen is appropriately selected during the analysis of the specimen. You can also select and fix.

  The selective binding substance (for example, nucleic acid) that can be immobilized on the upper surface of the convex portion can be appropriately selected as necessary as data, but may be a simple selective binding substance. Further, it is not necessary to bind the selective binding substance to all the upper surfaces of the convex portions, and the upper surfaces of the convex portions which are not fixed may be provided.

  In the substrate constituting the analysis chip of the present invention, when the selective binding substance is immobilized on the upper surface of the convex portion, the area of the upper surface of the convex portion is preferably substantially the same. Since the area of the upper surface of the convex portion is substantially the same, the area of the portion where the various selective binding substances are immobilized can be made the same, which is advantageous for later analysis. Here, the area of the upper part of the convex part is substantially the same means that the value obtained by dividing the largest upper surface area in the convex part by the smallest upper surface area is 1.2 or less.

The area of the upper surface of the convex portion on which the selective binding substance is immobilized is not particularly limited, but is 10 μm ² from the viewpoint that the amount of the selective binding substance can be reduced and handling is easy. As described above, it is preferably 1 mm ² or less, more preferably 300 μm ² or more and 0.8 mm ² or less.

  In the substrate constituting the analysis chip of the present invention, more preferably, the periphery of the surface having the region where the selective binding substance is immobilized on the substrate is further flat with substantially the same height as the upper end of the convex portion of the concave and convex portion. It is preferable that it is enclosed by the part. With such a structure, it becomes easy to apply the solution containing the specimen to the uneven portion, and the fine particles for stirring can be held in the recess without contacting the selective binding substance.

  It is preferable that the height of the upper surface of the convex part of the uneven part and the height of the flat part are substantially the same. That is, the difference between the height of the flat portion and the height of the upper surface of the convex portion is preferably 50 μm or less. If the difference between the height of the upper surface of the convex part and the height of the flat part exceeds 50 μm, the detectable fluorescence intensity may become weak, which is not preferable. The difference between the height of the flat portion and the height of the upper surface of the convex portion is more preferably 30 μm or less, and most preferably, the height of the flat portion and the height of the convex portion are the same.

  The height of the convex portion in the concave and convex portion of the substrate preferably used in the analysis chip of the present invention, that is, the difference in height between the upper surface of the convex portion and the bottom surface of the concave portion is preferably 10 μm or more and 500 μm or less, and 50 μm or more and 300 μm or less. More preferred. If the height of the convex portion is lower than 10 μm, a non-specifically adsorbed specimen sample other than the spot may be detected, and as a result, the S / N may be deteriorated. On the other hand, when the height of the convex portion is higher than 500 μm, problems such as the convex portion being easily broken and broken may occur, which is not preferable.

  Specific examples of the substrate constituting the analysis chip of the present invention are illustrated in FIGS.

  In the example shown in FIGS. 3 and 4, the surface of the substrate 1 is constituted by a concavo-convex portion 12 including a plurality of convex portions 11, and a flat portion 13 is provided around the concavo-convex portion 12. A selective binding substance (for example, nucleic acid) is immobilized on the upper surface of the convex portion 11. By using this flat portion, it becomes possible to easily focus the light beam for measurement such as the excitation light of the scanner on the upper surface of the convex portion.

  The material of the substrate of the analysis chip of the present invention is not particularly limited, but glass, ceramic, silicone resin, polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS) elastomer, etc. Examples include silicone rubber. Among these, polymethyl methacrylate, polystyrene, polydimethylsiloxane (PDMS) elastomer, glass, or silicone resin can be preferably used.

  The analysis chip substrate of the present invention is preferably at least partially black. By doing so, autofluorescence from the substrate can be reduced. The blackened portion of the substrate may be the substrate body provided with the concavo-convex portions, or may be a hydrophobic material or insulating layer provided on the side surfaces of the convex portions or the concave portions, or all of them.

  The substrate constituting the analysis chip of the present invention can be produced by various production methods. For example, when the material is a polymer or the like, it can be molded by an injection molding method, a hot embossing method, a method of polymerizing in a mold, or the like. Further, when the material is an inorganic substance such as glass or ceramic, it can be molded by a sandblast method, and when it is a silicone resin, it can be molded by a known semiconductor process.

  The molded substrate can be subjected to various surface treatments as necessary before immobilizing the selective binding substance on the surface. Specific examples of such surface treatment include those described in JP-A No. 2004-264289.

  In the present invention, an analysis chip refers to a chip used to apply a solution containing a sample to the chip and measure the presence / absence of the sample, the amount of the sample, the properties of the sample, and the like. Specifically, a biochip that measures the amount of the specimen and the presence or absence of the specimen by a reaction between the selective binding substance immobilized on the substrate surface and the specimen is mentioned. More specifically, a DNA chip having a nucleic acid immobilized on the substrate surface, a protein chip having a protein typified by an antibody immobilized on the substrate surface, a sugar chain chip having a sugar chain immobilized on the substrate surface, and a substrate surface Examples thereof include a cell chip on which cells are immobilized.

  In the present invention, the selective binding substance means various substances that can selectively bind to a specimen directly or indirectly. Typical examples of the selective binding substance that can bind to the surface of the substrate include nucleic acids, proteins, peptides, saccharides, and lipids.

  The nucleic acid may be DNA or RNA, or PNA. A single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a single-stranded nucleic acid having a base sequence complementary to the base sequence or a part thereof. Falls under the category of

  The nucleic acid may be derived from natural products such as living cells, or may be synthesized by a nucleic acid synthesizer. Preparation of DNA or RNA from living cells can be carried out by a known method, for example, the method of Blin et al. (Blin et al., Nucleic Acids Res. 3: 2303 (1976)) or the like for DNA extraction. Extraction can be performed by the method of Favaloro et al. (Favaloro et al., Methods Enzymol. 65: 718 (1980)) and the like. Examples of the nucleic acid to be immobilized include linear or circular plasmid DNA and chromosomal DNA, DNA fragments obtained by digesting these with restriction enzymes or chemically, DNA synthesized with enzymes in a test tube, or chemically synthesized oligos. Nucleotides and the like can also be used.

Examples of the protein include an antibody, an antigen-binding fragment of an antibody such as a Fab fragment or F (ab ′) ₂ fragment, and various antigens. Since an antibody or an antigen-binding fragment thereof selectively binds with a corresponding antigen, and the antigen selectively binds with a corresponding antibody, it corresponds to a “selective binding substance”.

  Examples of the saccharide include various monosaccharides and sugar chains such as oligosaccharides and polysaccharides.

  The lipid may be a simple lipid or a complex lipid.

  Furthermore, antigenic substances other than the nucleic acids, proteins, saccharides and lipids can also be immobilized. In addition, as a selective binding substance, cells may be immobilized on the surface of the substrate.

  The analysis chip of the present invention further includes a cover member that covers the surface of the substrate and is bonded to the substrate. By providing the cover member, the solution containing the specimen can be easily hermetically sealed, and as a result, the selective binding substance immobilized on the specimen and the region of the substrate (12 in FIG. 3 or FIG. 4). The reaction with can be carried out stably. Moreover, fine particles can be injected (stored) in advance into the analysis chip of the present invention, and the work of applying the sample solution can be easily performed. Further, even in the operation of closing the through hole after applying the sample solution, there is an advantage that the background noise does not increase because the tape and the sealant do not come into contact with the sample solution.

  FIG. 5 is a perspective view showing an example of a schematic aspect of the analysis chip of the present invention further including a cover member, an adhesive member, a through hole, and a liquid level holding chamber in addition to the substrate.

  The cover member may be bonded so as to cover a part of at least one surface of the substrate and to have a gap between the substrate and the cover member. The substrate preferably has a selective binding substance immobilized on a region of the substrate located in the void. That is, preferably, the cover member is bonded to the substrate so that the region where the selective binding substance is immobilized exists in the gap. The cover member may be bonded in any manner as long as the gap is formed, but is preferably bonded via an adhesive member such as a double-sided tape or a resin composition.

  The cover member may have one or more through holes communicating with the gap, and preferably has a plurality of through holes. More specifically, it is preferable that there are a plurality of through-holes with respect to one gap, and among these, it is particularly preferable that the number of through-holes is 3 to 6 because the solution containing the specimen can be easily filled. As will be described later, when the gap is divided into a plurality of spaces that do not communicate with each other, it is preferable that each space has a plurality, more preferably 3 to 6, through holes. When the cover member has a plurality of through holes, the diameters thereof may be the same or different, but one of the plurality of through holes functions as an apply port and the other through hole functions as an air outlet. In this case, from the viewpoint of easy application of the sample solution and hermetic retention of the solution, it is preferable that only the apply port has a wide hole diameter necessary for the application and other through holes have a narrower hole diameter. Specifically, the through hole size of the apply port is preferably in the range of 0.01 mm to 2.0 mm as described above, and the diameters of the other through holes are preferably 0.01 mm to 1.0 mm.

  Such a cover member is preferably detachably bonded to the substrate. When the analysis chip of the present invention is used as a DNA chip, it is usually necessary to read the DNA chip with a dedicated scanner. However, in the state where the cover member is adhered, it is difficult to set in the dedicated scanner. However, when the scanning operation is performed, the cover member and the optical parts of the scanner may come into contact with each other, which may cause a failure. Further, even if reading through the cover member is possible, the reading value may be inaccurate. Therefore, it is preferable that the cover member is removable so that the cover member can be removed in the reading step.

  A mode in which the cover member is detachably attached to the substrate is not particularly limited, but a mode in which the cover member and the substrate can be detached without being damaged is preferable. For example, it can adhere | attach via adhesive members, such as a double-sided tape and a resin composition.

  The shape of the cover member is not particularly limited as long as it covers at least a part of the surface of the substrate and can be bonded so as to have a gap between the substrate and the cover member. A structure having a protruding portion in a portion far from the substrate than a portion close to the substrate, that is, an overhang structure may be provided. Providing an overhang structure is preferable because it is easy to remove the cover member without damaging the substrate.

  The material of the cover member constituting the analysis chip of the present invention is not particularly limited, but a transparent material is preferable so that the state of the solution can be observed when the sample solution is applied. Such materials include glass or plastic. In particular, transparent resins such as polystyrene, polymethyl methacrylate, and polycarbonate can be preferably used from the viewpoint that structures such as through holes and liquid level holding chambers can be easily produced. The method for producing the cover member is not particularly limited, and processing by cutting or injection molding is possible. From the viewpoint that mass production is possible, an injection molding method can be preferably used.

  A preferred example of the relationship between the concavo-convex portion, the cover member, and the fine particles in the analysis chip of the present invention will be described with reference to FIG. In the example shown in FIG. 6, the selective binding substance 45 such as DNA is immobilized on the upper surface of the convex portion 11 of the substrate 1. The fine particles (in this case, spherical beads) 2 are placed in the gaps in the recesses of the substrate 1. The selective binding substance 45 and the fine particles 2 come into contact with a solution (not shown) containing the specimen. The sample solution is held in a gap defined by the substrate 1, the adhesive member 30 and the cover member 3. In the example of FIG. 6, the shortest distance between the upper surface of the convex portion 11 of the substrate and the cover member 3 is less than the diameter of the fine particles 2. Thereby, the fine particles can no longer contact the upper surface of the convex portion 11, and it is possible to prevent the selective binding substance 45 on the upper surface of the convex portion 11 from being damaged. When the fine particles have an aspherical shape such as an ellipse, for example, if the shortest distance between the upper surface of the convex portion and the container is less than the minimum diameter of the fine particles, the contact between the upper surface of the convex portion 11 and the fine particles is similarly prevented. It is possible to prevent the selective binding substance 45 from being damaged.

  Such an analysis chip of the present invention can be used for analysis of various samples. That is, a sample is brought into contact with the substrate on which the selective binding substance of the analysis chip of the present invention is immobilized to selectively bind to the selective binding substance, and is bonded to the substrate via the selective binding substance. A sample can be analyzed by measuring the presence or absence or amount of the sample.

  In the sample analysis method, first, a sample is brought into contact with and selectively bound to a substrate on which a selective binding substance constituting the analysis chip of the present invention is immobilized. That is, a solution in which a sample having the above-described labeling, amplification, etc. applied to the substrate is dissolved in an aqueous solution or an appropriate buffer solution (sometimes referred to as “sample solution” in this specification) is brought into contact with the substrate. It ’s fine.

  The contact with the substrate to which the selective binding substance is bound can be performed by making the specimen into a solution such as an aqueous solution or an appropriate buffer solution and injecting it into the uneven portion of the substrate with a normal instrument such as a pipette. The sample can be applied from the through hole by, for example, injecting it from the through hole with a normal instrument such as a pipette.

  Affixing of the sealing member to the cover member can be performed in a mode in which part or all, preferably all, of the through hole is sealed. Preferred examples of the sealing member include a flexible tape such as an adhesive tape made of polyimide film such as Kapton (registered trademark, manufactured by Toray DuPont), polyester, cellophane, or vinyl chloride. However, the present invention is not limited to this, and any non-flexible plate-like adhesive-bondable member can be used, and an atypical sealing agent can also be used. From the viewpoint of obtaining the effect of the present invention by the liquid level chamber more favorably, a flexible tape and a plate-like member are preferable, and from the viewpoint of ease of operation, a flexible tape is further provided. preferable.

  A specific example of sealing will be described with reference to FIG. 6 again. In the example of FIG. 6, after applying a sample solution (not shown) from the through hole 32, a flexible adhesive tape 34 is attached as a sealing member so as to cover the entire surface of the liquid level holding chamber 33. The through hole is sealed. According to such an embodiment, it is possible to achieve sealing that is simple and does not cause leakage of the sample solution or measurement error.

  In the specimen analysis method, the selective binding means that the selective binding substance interacts with the specimen, and the specimen is bound to the substrate on which the selective binding substance is immobilized via the selective binding substance. It means that In the case of the analysis chip of the present invention, by selectively swinging and rotating the chip, the microparticles move in the sample solution by weight, vibration, and centrifugal force, so that selective binding can be promoted efficiently.

  The reaction temperature and time for the selective binding of the selective binding substance and the specimen are appropriately selected according to the nucleic acid chain length of the specimen to be hybridized, the type of antigen and / or antibody involved in the immune reaction, etc. However, in the case of nucleic acid hybridization, it is usually about 40 ° C. to 70 ° C. for about 1 minute to several tens of hours, and in the case of an immune reaction, it is usually about room temperature to about 50 ° C. for about 1 minute to several hours. . Further, if necessary, the selective binding can be promoted by swinging, rotating, etc. the substrate on which the selective binding substance is immobilized.

  The analysis chip of the present invention can stir the sample solution efficiently by moving the fine particles in the analysis chip. As means for moving the fine particles, preferably the method of rotating the analysis chip to drop the fine particles in the direction of gravity, the method of setting the analysis chip containing the fine particles in a shaker and shaking or swinging the substrate, The method of moving fine particles by magnetic force using magnetic fine particles is used, but the method of setting the chip on a shaker and rotating it in a horizontal plane has a large moving range of fine particles, and the result is that it moves without bias. It is preferably used because the liquid can be efficiently stirred. At this time, the rotational speed of the turning rotation is preferably 10 to 1000 rotations / minute, and more preferably 100 to 500 rotations / minute.

  After completion of the selective bonding, the cover member is usually detached and can be used for the next step.

  In the analysis method, after the selective binding described above, the mass of the analyte bound on the substrate via the selective binding substance is measured. This measurement can also be performed in exactly the same manner as in the conventional analysis chip. For example, the mass of a sample that is appropriately fluorescently labeled and bound to a selective binding substance can be measured by reading the amount of fluorescence with a known scanner or the like.

  In the specimen analysis method, when a nucleic acid is immobilized as a selective binding substance, a nucleic acid having a sequence complementary to this nucleic acid or a part thereof can be measured. When an antibody or antigen is immobilized as a selective binding substance, the antigen or antibody that immunoreacts with this antibody or antigen can be measured. Note that “measurement” in this specification includes both detection and quantification.

  The present invention further relates to an analysis kit. The analysis kit here is for constituting the analysis chip of the present invention, and includes at least a substrate having a selective binding substance immobilized on the surface thereof, and a cover member bonded to the substrate. An analysis chip having a gap between the substrate and the cover member, in which fine particles are movably stored in the gap, and a reaction solution for the binding reaction between the selective binding substance and the analyte. A reaction solution containing an antifoaming agent and / or a nonionic surfactant is included.

  In addition to the above, what can be included in the analysis kit of the present invention includes, but is not limited to, a sealing seal, an application tip, an application stand, a cotton swab, and the like.

  As a method for using the analysis kit of the present invention, it can be used for detection of a sample that selectively binds to a selective binding substance immobilized on an analysis chip. Specifically, the sample is labeled, mixed with the above-mentioned “reaction solution”, and this is injected into the analysis chip from the through hole provided in the cover member bonded to the substrate. After the injection is completed, the through-hole of the analysis chip is closed with a sealing seal, the selective binding substance on the surface of the specimen and the chip substrate for analysis is reacted, and the label of the specimen is detected to detect the selective binding substance and the specific substance. An analyte that binds to can be detected.

  The invention is illustrated in more detail by the following examples. The present invention is not limited to the following examples.

Comparative Example 1
(1) Fabrication of analysis chip substrate Polymethylmethacrylate having a shape as described below by injection molding is prepared by using a known method LIGA (Lithographie Galvanforming Abform) process. A substrate made of PMMA) was obtained. The average molecular weight of PMMA used was 50,000, and carbon black (# 3050B manufactured by Mitsubishi Chemical) was contained in PMMA at a ratio of 1% by weight to make the substrate black. When the spectral reflectance and spectral transmittance of this black substrate were measured, the spectral reflectance was 5% or less at any wavelength in the visible light region (wavelength 400 nm to 800 nm), and in the same range of wavelengths, The transmittance was 0.5% or less. Neither spectral reflectance nor spectral transmittance had a specific spectral pattern (such as a peak) in the visible light region, and the spectrum was uniformly flat. Note that the spectral reflectance is a spectrum obtained when specularly reflected light from a substrate is captured using an apparatus (manufactured by Minolta Camera, CM-2002) equipped with an illumination / light-receiving optical system conforming to the condition C of JIS Z 8722. The reflectance was measured.

  As the substrate, the substrate having the shape illustrated in FIGS. 3 and 4 has an outer shape of 76 mm in length, 26 mm in width, and 1 mm in thickness, and 39.4 mm in length, 19.0 mm in width, deep in the center of the substrate. A recess having a thickness of 0.15 mm (corresponding to the recess 10 in FIG. 3) is provided, and 9248 projections having a diameter of 0.1 mm and a height of 0.15 mm (corresponding to the protrusion 11 in FIG. 3) are provided in the recess. The provided substrate (hereinafter referred to as “substrate A”) was used. In this substrate A, the difference in height between the upper surface of the convex portion (corresponding to the convex portion 11 in FIG. 3) and the upper surface of the flat portion (corresponding to the convex portion 13 in FIG. 3) (the convex portion is the average height) is It was 3 μm or less. Further, the variation in the height of the upper surface of the convex portion (corresponding to the convex portion 13 in FIG. 3) (the difference between the height of the upper surface of the highest convex portion and the height of the upper surface of the lowest convex portion) was 3 μm or less. Further, the pitch of the convex portions (L1 in FIG. 4; the distance from the central portion of the convex portion to the central portion of the adjacent convex portion) was set to 0.5 mm.

  The substrate A was immersed in a 10N aqueous sodium hydroxide solution at 70 ° C. for 12 hours. This was washed in the order of pure water, 0.1N HCl aqueous solution, and pure water to generate carboxyl groups on the substrate surface.

(2) Immobilization of selective binding substance Oligonucleotides were immobilized on the substrate A as selective binding substances (probe DNAs) under the following conditions. As the oligonucleotide, an oligonucleotide set “Homo sapiens (human) AROS V4.0 (60 bases each)” for DNA microarray manufactured by Operon was used. This oligonucleotide was dissolved in pure water to a concentration of 0.3 nmol / μL to obtain a stock solution. When spotting this stock solution on the substrate, PBS (8 g NaCl, 2.9 g Na ₂ HPO ₄ · 12H ₂ O, 0.2 g KCl, and 0.2 g KH ₂ PO ₄ was added. In addition, it is dissolved in pure water, diluted to 1 L, adjusted to pH 5.5 by adding hydrochloric acid, and diluted 10-fold to make the final concentration of probe DNA 0.03 nmol / μL, and PMMA In order to condense the carboxyl group generated on the substrate surface and the terminal amino group of the probe DNA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added, and the final concentration was 50 mg / mL. did. This solution was spotted on the upper surfaces of all the convex portions of the substrate A using an arrayer (spotter) (manufactured by Nippon Laser Electronics; Gene Stamp-II). The spotted substrate was then placed in a sealed plastic container and incubated for about 20 hours at 37 ° C. and 100% humidity. Finally, the substrate was washed with pure water, and dried by centrifuging with a spin dryer.

(3) Affixing the cover member to the analysis chip substrate A cover member was affixed to the substrate A on which the selective binding substance was immobilized as follows. As the cover member, a PMMA flat plate having a length of 41.4 mm, a width of 21 mm, and a thickness of 1 mm was produced by cutting to obtain a cover member. The produced cover member was provided with a through hole and a liquid level holding chamber as illustrated in 32 and 33 of FIG. Then, a double-sided tape having a length of 41.4 mm and a width of 21 mm is used as an adhesive member, and the double-sided tape is laminated with a thickness of 50 μm so as to border the cover member. Attached to A.

(4) Preparation of specimen As a specimen, aRNA (antisense RNA), which is common as a specimen of a microarray, was used. 5 μg of Cy3-labeled aRNA was obtained from 5 μg of commercially available human cell-derived total RNA (Human Reference RNA manufactured by CLONTECH) using an Ambion aRNA preparation kit. (5) High of selective binding substance and specimen Reaction solution for hybridization Unless otherwise specified in the following Examples and Comparative Examples, the labeled aRNA prepared above was mixed with 1% by weight BSA, 5 × SSC, 0.01% by weight salmon sperm DNA, 0.1% by weight. A solution diluted with a solution of% SDS (each concentration is a final concentration) was used.

(6) Injection (storage) of fine particles into analysis chip
120 mg of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm are formed on the substrate A to which the cover member is attached in the above (3), and a void formed by the substrate A and the cover member (the uneven structure on the surface of the substrate A). It was injected (stored) into the recess. The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was designated as “Analysis chip 1”.

  As a sample, aRNA (antisense RNA), which is common as a sample of a microarray, was used. 5 μg of Cy3-labeled aRNA was obtained from 5 μg of commercial human cell-derived total RNA (Human Reference RNA manufactured by CLONTECH) using an aRNA preparation kit manufactured by Ambion.

(7) Evaluation of hybridization reaction and number of bubbles generated Using a micropipette, 165 μL of a reaction solution containing 200 ng of Cy3-labeled aRNA was injected from the through-hole into the space (reaction tank) between the substrate A of the analysis chip 1 and the cover member. did. At this time, the solution could be easily injected, and bubbles were not mixed. Using Kapton tape (As One) as a sealing material, four through holes were closed. A hybridization chamber (Takara Hybridization chamber (manufactured by Takara Bio Inc.)) is closely attached to a sheet shaking table (MMS FIT-S, manufactured by Tokyo Rika Kikai Co., Ltd.), and the analysis chip 1 is placed in the hybridization chamber. Set. At this time, 15 μL of ultrapure water was dropped into the dents at both ends of the position where the analysis chip 1 is set. After closing the hybridization chamber lid, the six fixing screws were tightened and fixed, and a shaker (Tokyo Rika Instrument (Tokyo Rika Instrument Co., Ltd. FMS-1000)) set in a constant temperature chamber set to 42 ° C. The product was placed on MMS-310) and was fixed. The front surface of the thermostatic chamber was shielded from light with aluminum foil, and incubated at 42 ° C. for 16 hours while swirling at 250 rpm. After the incubation, the analysis chip 1 was taken out from the hybridization chamber.

  Through the cover member, the number of bubbles in the reaction solution observed on the substrate A of the analysis chip 1 was counted. As a result of carrying out the hybridization reaction using the analysis chip 1 10 times, the number of bubbles generated in the reaction solution was 13.0 on average (Table 1).

(8) Measurement of fluorescence signal value and evaluation of variation in detection sensitivity After the cover member and the double-sided tape adhered to the substrate A of the analysis chip 1 were removed, the substrate A was washed and dried. A labeled body of a sample that has undergone a hybridization reaction in a state where the substrate A after the above processing is set on a DNA chip scanner (GenePix 4000B manufactured by Axon Instruments), the laser output is 33%, and the voltage setting of the photomultiplier is 500. Signal value (fluorescence intensity) and background noise were measured. Of the 9248 spots, 32 were used as negative control spots for background fluorescence measurement, and the true signal value of each spot was calculated by subtracting the background signal value from each signal value.

  As an evaluation of the variation in detection sensitivity due to uneven reaction of the hybridization reaction due to the appearance of bubbles, the hybridization reaction using the analysis chip 1 was performed 10 times, and the background signal value variation (CV value = all times) at each time. The standard deviation of the background signal value / the average value (%) of all background signal values was calculated. As a result, the average value of the variation (CV value) of the background signal value in 10 evaluations was 12.1% (Table 1).

Comparative Example 2
Evaluation using the analysis chip 1 produced in the same manner as in Comparative Example 1 was performed, except that the reaction solution prepared in Comparative Example 1 (5) was degassed as follows. 175 μl of the reaction solution was placed in a 0.2 ml PCR tube (72.7737.002 manufactured by Assist), and set in a deaeration device (ULVAC Aspirator NDA-015 type) with the lid opened to perform deaeration. The ultimate pressure during degassing was 50 hPa on the display of the device, and the degassing time was 25 minutes.

When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 9.0 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Reference Example 1 (8), it was 10.5% (Table 1).

Reference example 1
To the reaction solution prepared in Comparative Example 1 (5), an antifoaming agent “PE-L” manufactured by Wako Pure Chemical Industries, Ltd. was added as an antifoaming agent so that the v / v concentration was 0.05%. Evaluation was performed using the analytical chip 1 produced in the same manner as in Comparative Example 1 except that the deaeration treatment was performed as follows. 175 μl of the reaction solution was placed in a 0.2 ml PCR tube (72.7737.002 manufactured by Assist), and set in a deaeration device (ULVAC Aspirator NDA-015 type) with the lid opened to perform deaeration. The ultimate pressure during degassing was 50 hPa on the device display, and the degassing time was 25 minutes.

When the number of bubbles in the reaction solution observed on the substrate after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the number of bubbles was 10 times. The number was 1.2 (Table 1). Further, the variation (CV value) in the background signal value was calculated in the same manner as in Reference Example 1 (8), and it was 6.9% (Table 1).

Reference example 2
Degassing analysis in the same manner as in Reference Example 1 except that Wako Pure Chemical Industries, Ltd. antifoaming agent “SI” was added to the reaction solution to a 0.05% v / v concentration. An evaluation using the chip 1 was carried out. The deaeration time was 25 minutes.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 2.9 (Table 1). Further, the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), and it was 7.3% (Table 1).

Reference example 3
Analytical chip degassed in the same manner as in Reference Example 1 except that “glycolic acid” manufactured by Wako Pure Chemical Industries, Ltd. was added to the reaction solution to a 0.05% v / v concentration. Evaluation using 1 was carried out. The deaeration time was 25 minutes.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 4.7 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 7.8% (Table 1).

Example 4
120 mg of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm coated with the surfactant sodium dodecyl sulfate (SDS) on the substrate A on which the cover member was attached in Comparative Example 1 (3), the substrate A and the cover member Were injected (stored) in the voids formed in the above (recesses of the concavo-convex structure on the surface of the substrate A), and the other analysis chips were prepared in the same manner as in Example 1. The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was referred to as “analysis chip 2”. The reaction solution was adjusted in the same manner as in Reference Example 1.

  As the fine particles coated with SDS, 10 g of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm were placed in a stainless steel vat (10 cm × 10 cm × 5 cm), and 50 ml of 0.1% SDS aqueous solution was added. After sonicating this for 10 minutes, the supernatant (SDS component) was removed, and the fine particles were dried at 70 ° C. for 12 hours using an oven. In addition, when the surface roughness of the microparticles | fine-particles before a process was measured, the centerline average roughness (Ra value) of the surface was 165 nm. The Ra value on the surface of the fine particles was measured with a scanning electron microscope (Model ESA-2000, manufactured by Elionix Co., Ltd.) after the surface was vacuum-deposited with Au. The centerline average roughness was measured for 10 arbitrary samples with an observation magnification of 10,000 times and a cutoff value of 0, and the average value was obtained.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.3 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.2% (Table 1).

Example 5
Substrate A was coated with 120 mg of zirconia fine particles having a diameter of 180 μm obtained by coating the substrate A on which the cover member was attached in Comparative Example 1 (3) with the surfactant polyoxyethylene (20) sorbitan monolaurate (polysorbate 20, Tween 20). And a cover member were injected (stored) into the gap formed by the concave portion of the concave-convex structure on the surface of the substrate A, and the analysis chip was produced in the same manner as in Example 1. The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was referred to as “analysis chip 3”. The reaction solution was adjusted in the same manner as in Reference Example 1.

  The fine particles coated with Tween 20 were processed in the same procedure as in Example 4, and then washed once with 400 mL of deionized water (Milli Q water) and dried at 70 ° C. for 4 hours.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 1.3 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.5% (Table 1).

Example 6
In the substrate A and the cover member, 120 mg of zirconia fine particles having a diameter of 180 μm coated with the surfactant cetyltrimethylammonium bromide (CTAB) on the substrate A on which the cover member was attached in Comparative Example 1 (3). A chip for analysis was prepared in the same manner as in Reference Example 1 except that it was injected (stored) into the formed void (the concave portion of the concave-convex structure on the surface of the substrate A). The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was referred to as “analysis chip 4”. The reaction solution was adjusted in the same manner as in Reference Example 1.

  The fine particles coated with CTAB were processed in the same procedure as in Example 4, and then washed once with 400 mL of deionized water (Milli-Q water) and dried at 70 ° C. for 4 hours.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 3.1 (Table 1). Further, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 7.7% (Table 1).

Example 7
The “analysis chip 2” was used in the same manner as in Example 4, and the reaction solution was used with the surfactant SDS concentration contained in the reaction solution of Reference Example 1 increased from 0.1% to 2%.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.2 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.1% (Table 1).

Reference Example 8
Comparative Example 1 except that the defoaming agent “PE-L” manufactured by Wako Pure Chemical Industries, Ltd. was added to the reaction solution prepared in Comparative Example 1 (5) so that the v / v concentration was 0.05%. Evaluation using the analysis chip 1 produced in the same manner as described above was performed.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 6.2 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 8.7% (Table 1).

Example 9
The “analysis chip 2” was used in the same manner as in Example 4, and the reaction solution used was obtained by changing the surfactant contained in the reaction solution of Comparative Example 2 from 0.1% SDS to 0.1% Tween 20. .

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.7 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 7.0% (Table 1).

Example 10
The “analysis chip 2” was used in the same manner as in Examples 4 and 8, and the reaction solution used was obtained by changing the surfactant contained in the reaction solution of Comparative Example 2 from 0.1% SDS to 10% Tween 20. .

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.4 (Table 1). Moreover, when the variation (CV value) of the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.6% (Table 1).

Example 11
The “analysis chip 3” was used in the same manner as in Example 5, and the reaction solution used was obtained by changing the surfactant contained in the reaction solution of Comparative Example 2 from 0.1% SDS to 0.1% Tween 20. .

  When the number of generated bubbles after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.5 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.3% (Table 1).

Example 12
The “analysis chip 3” was used in the same manner as in Examples 5 and 10, and the reaction solution was changed from 0.1% SDS to 10% Tween 20 for the surfactant contained in the reaction solution of Comparative Example 2. .

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.4 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.1% (Table 1).

Example 13
120 mg of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm coated with the surfactant Tween 60 are formed on the substrate A on which the cover member is pasted in Comparative Example 1 (3). A chip for analysis was prepared in the same manner as in Reference Example 1 except that it was injected (stored) into a void (a concave portion of the concave-convex structure on the surface of the substrate A). The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was referred to as “analysis chip 5”.

  As fine particles coated with Tween 60, 10 g of zirconia fine particles having a diameter of 180 μm (manufactured by Toray Industries, Inc.) were placed in a stainless steel vat (10 cm × 10 cm × 5 cm), and 50 ml of 5% Tween 60 aqueous solution was added. After sonicating this for 10 minutes, the supernatant (Tween 20 component) was removed, and the fine particles were dried at 70 ° C. for 12 hours using an oven.

  As the reaction solution, the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% Tween 60.

  In the same manner as in Comparative Example 1 (7), the number of bubbles generated after the hybridization reaction was counted, and the average of 10 evaluations was found to be 0.6 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.3% (Table 1).

Example 14
The “analysis chip 5” was used in the same manner as in Example 13, and the reaction solution used was obtained by changing the surfactant contained in the reaction solution of Comparative Example 2 from 0.1% SDS to 10% Tween 60.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.4 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.1% (Table 1).

Example 15
120 mg of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm, coated with the surfactant Tween 80, are formed on the substrate A and the cover member, which are coated with the cover member A in Comparative Example 1 (3). A chip for analysis was prepared in the same manner as in Reference Example 1 except that it was injected (stored) into a void (a concave portion of the concave-convex structure on the surface of the substrate A). The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was referred to as “analysis chip 6”.

  As fine particles coated with Tween 80, 10 g of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm were placed in a stainless steel vat (10 cm × 10 cm × 5 cm), and 50 ml of 5% Tween 80 aqueous solution was added. After sonicating this for 10 minutes, the supernatant (Tween 80 component) was removed, and the fine particles were dried at 70 ° C. for 12 hours using an oven.

  As the reaction solution, the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% Tween 80.

  In the same manner as in Comparative Example 1 (7), the number of bubbles generated after the hybridization reaction was counted, and the average of 10 evaluations was found to be 0.5 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.3% (Table 1).

Example 16
The “analysis chip 6” was used in the same manner as in Example 15, and the reaction solution used was obtained by changing the surfactant contained in the reaction solution of Comparative Example 2 from 0.1% SDS to 10% Tween 80.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.4 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 5.9% (Table 1).

Example 17
As in Examples 4 and 9, “Analytical chip 2” was used, and for the reaction solution, the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% Pluronic F-68. I used something.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.9 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.8% (Table 1).

Example 18
120 mg of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm coated with the surfactant Pluronic F-68 on the substrate A on which the cover member was attached in Comparative Example 1 (3), were formed with the substrate A and the cover member. A chip for analysis was prepared in the same manner as in Reference Example 1 except that it was injected (stored) into the voids (recesses of the concavo-convex structure on the surface of the substrate A). The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was designated as “analysis chip 7”.

  The fine particles coated with Pluronic F-68 were charged with 10 g of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm in a stainless steel vat (10 cm × 10 cm × 5 cm), and 50 ml of 3% Pluronic F-68 aqueous solution was added. After sonicating this for 10 minutes, the supernatant (Pluronic F-68 component) was removed, and the fine particles were dried at 70 ° C. for 12 hours using an oven.

  As for the reaction solution, the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% Pluronic F-68.

  In the same manner as in Comparative Example 1 (7), the number of bubbles generated after the hybridization reaction was counted, and the average of 10 evaluations was found to be 0.7 (Table 1). Moreover, when the variation (CV value) of the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.8% (Table 1).

Example 19
The “analysis chip 7” was used in the same manner as in Example 15, and the reaction solution used was obtained by changing the surfactant contained in the reaction solution of Comparative Example 2 from 0.1% SDS to 3% Pluronic F-68. .

  When the number of generated bubbles after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.5 (Table 1). Moreover, when the variation (CV value) of the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.8% (Table 1).

Example 20
“Analysis chip 2” was used in the same manner as in Examples 4 and 8, and the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% Pluronic F-127 for the reaction solution. I used something.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.7 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.5% (Table 1).

Example 21
120 mg of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm coated with the surfactant Pluronic F-127 on the substrate A on which the cover member was attached in Comparative Example 1 (3), were mixed between the substrate A and the cover member. A chip for analysis was prepared in the same manner as in Reference Example 1 except that it was injected (stored) into the formed void (the concave portion of the concave-convex structure on the surface of the substrate A). The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was designated as “analysis chip 8”.

  The fine particles coated with Pluronic F-127 were charged with 10 g of zirconia fine particles having a diameter of 180 μm (manufactured by Toray Industries, Inc.) in a stainless steel vat (10 cm × 10 cm × 5 cm), and 50 ml of 3% Pluronic F-127 aqueous solution was added. After sonicating this for 10 minutes, the supernatant (Pluronic F-127 component) was removed, and the microparticles were dried at 70 ° C. for 12 hours using an oven.

  As the reaction solution, the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% Pluronic F-127.

  In the same manner as in Comparative Example 1 (7), the number of bubbles generated after the hybridization reaction was counted, and the average of 10 evaluations was found to be 0.8 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.7% (Table 1).

Example 22
The “analysis chip 8” was used in the same manner as in Example 21, and the reaction solution used was obtained by changing the surfactant contained in the reaction solution of Comparative Example 2 from 0.1% SDS to 3% Pluronic F-127. did.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 0.4 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.3% (Table 1).

Example 23
“Analysis chip 2” was used in the same manner as in Examples 4 and 9, and the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% Triton X-100 for the reaction solution. I used something.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 1.6 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.0% (Table 1).

Example 24
120 mg of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm coated with the surfactant Triton X-100 on the substrate A on which the cover member was attached in Comparative Example 1 (3) A chip for analysis was prepared in the same manner as in Reference Example 1 except that it was injected (stored) into the formed void (the concave portion of the concave-convex structure on the surface of the substrate A). The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was referred to as “analysis chip 9”.

  The fine particles coated with Triton X-100 were charged with 10 g of zirconia fine particles having a diameter of 180 μm (manufactured by Toray Industries, Inc.) in a stainless steel vat (10 cm × 10 cm × 5 cm), and 50 ml of 5% Pluronic F-127 aqueous solution was added. . After sonicating this for 10 minutes, the supernatant (Pluronic F-127 component) was removed, and the microparticles were dried at 70 ° C. for 12 hours using an oven.

  As the reaction solution, the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% Triton X-100.

  In the same manner as in Comparative Example 1 (7), the number of bubbles generated after the hybridization reaction was counted, and the average value of 10 evaluations was found to be 1.3 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 7.2% (Table 1).

Example 25
In the same manner as in Example 24, “Analysis chip 9” was used, and the reaction solution was changed from 0.1% SDS to 0.1% Triton X-100 as the surfactant contained in the reaction solution of Comparative Example 2. used.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 1.1 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 5.9% (Table 1).

Example 26
“Analysis chip 2” was used in the same manner as in Examples 4 and 9, and the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% NP-40 for the reaction solution. I used something.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 2.1 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 7.2% (Table 1).

Example 27
120 mg of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm, coated with surfactant NP-40, are formed on substrate A and the cover member on substrate A to which the cover member has been attached in Comparative Example 1 (3). A chip for analysis was prepared in the same manner as in Reference Example 1 except that it was injected (stored) into the voids (recesses of the concavo-convex structure on the surface of the substrate A). The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was designated as “analysis chip 10”.

  As for the fine particles coated with NP-40, 10 g of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm are placed in a stainless steel vat (10 cm × 10 cm × 5 cm), and 50 ml of 5% 0.1% NP-40 aqueous solution is placed. added. After sonicating this for 10 minutes, the supernatant (0.1% NP-40 component) was removed, and the microparticles were dried at 70 ° C. for 12 hours using an oven.

  As the reaction solution, the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% NP-40.

  In the same manner as in Comparative Example 1 (7), the number of bubbles generated after the hybridization reaction was counted, and the average of 10 evaluations was found to be 1.8 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.7% (Table 1).

Example 28
The “analysis chip 9” was used in the same manner as in Example 27, and the reaction solution used was obtained by changing the surfactant contained in the reaction solution of Comparative Example 2 from 0.1% SDS to 10% NP-40. .

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 1.6 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 6.9% (Table 1).

Comparative Example 3
120 mg of zirconia fine particles (manufactured by Toray Industries, Inc.) having a diameter of 180 μm coated with the surfactant CHAPSO are formed on the substrate A on which the cover member is attached in Comparative Example 1 (3). A chip for analysis was prepared in the same manner as in Reference Example 1 except that it was injected (stored) into a void (a concave portion of the concave and convex structure on the surface of the substrate A). The fine particles were injected from the through hole (the through hole 32 illustrated in FIGS. 1, 2, and 6) of the cover member. The analysis chip obtained as described above was designated as “analysis chip 11”.

  As fine particles coated with CHAPSO, 10 g of zirconia fine particles having a diameter of 180 μm (manufactured by Toray Industries, Inc.) were placed in a stainless steel vat (10 cm × 10 cm × 5 cm), and 50 ml of 1% CHAPSO aqueous solution was added. This was sonicated for 10 minutes, the supernatant (CHAPSO component) was removed, and the fine particles were dried at 70 ° C. for 12 hours using an oven.

  As the reaction solution, the surfactant contained in the reaction solution of Comparative Example 2 was changed from 0.1% SDS to 0.1% CHAPSO.

  In the same manner as in Comparative Example 1 (7), the number of bubbles generated after the hybridization reaction was counted, and the average value of 10 evaluations was found to be 2.0 (Table 1). Moreover, when the variation (CV value) in the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 9.0% (Table 1).

Comparative Example 4
The “analysis chip 11” was used in the same manner as in Comparative Example 3, and the reaction solution was obtained by changing the surfactant contained in the reaction solution of Comparative Example 2 from 0.1% SDS to 1% CHAPSO.

  When the number of bubbles generated after the hybridization reaction was counted in the same manner as in Comparative Example 1 (7), the average of 10 evaluations was 4.5 (Table 1). Moreover, when the variation (CV value) of the background signal value was calculated in the same manner as in Comparative Example 1 (8), it was 8.1% (Table 1).

Above Reference Example 1～3,8, Example 4～7,9 to 28, from the results of Comparative Examples 1 to 4, the reaction liquid of the tip and the specimen defoamer, or nonionic surfactant By containing, it is possible to reduce the variation of data (background signal CV value) by suppressing the generation of bubbles, and more effective by combining with degassing of sample solution and surfactant treatment of bead surface. It was found that the generation of bubbles can be suppressed.

  The present invention includes a substrate on which a selective binding substance that selectively binds to a specimen is immobilized, and an analysis chip that can stir a specimen solution with fine particles, suppressing variations in detection sensitivity and performing highly sensitive detection. It is possible. The present invention is particularly useful as an analysis chip for detection of various biological substances in the pharmaceutical and medical fields, and is also useful as an analysis chip for detection of trace substances in the food and environmental fields. is there.

FIG. 1 is a cross-sectional view schematically showing an example of a substrate on which a selective binding substance constituting the analysis chip of the present invention is immobilized. FIG. 2 is a cross-sectional view schematically showing an example in which bubbles are generated on the surface of the substrate by using the substrate of FIG. FIG. 3 is a perspective view schematically showing an example of a substrate on which a selective binding substance constituting the analysis chip of the present invention is immobilized. FIG. 4 is a cross-sectional view schematically showing an example of a substrate on which a selective binding substance constituting the analysis chip of the present invention shown in FIG. 3 is immobilized. FIG. 5 is a perspective view schematically showing an example of a preferable relationship between the uneven portion, the cover member, and the fine particles of the analysis chip of the present invention. FIG. 6 is a longitudinal sectional view schematically showing an example of a preferable relationship between the uneven portion, the cover member, and the fine particles of the analysis chip of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Substrate 2 Fine particle 3 Cover member 10 Concave part 11 Convex part 12 Area | region (uneven part) to which the selective binding substance was fixed
13 Flat part 26 Example of generated bubble 30 Adhesive member 30A Adhesive member 32 with partition structure Through hole 33 Liquid level chamber 34 Sealing member (tape)
45 Selective binding substance L1 immobilized on substrate L1 convex pitch

Claims (7)

A substrate having a selective binding substance immobilized on the surface thereof and a cover member bonded to the substrate, and having a gap between the substrate and the cover member, the fine particles can be movably stored in the gap. a analytical chips, surfactant on the surface of the fine particles are coated, a reaction solution for the coupling reaction of the selective binding substance and the analyte in the void of a nonionic surfactant An analysis chip for holding a reaction solution. The analysis chip according to claim 1 , wherein the surfactant coated on the surface of the fine particles is an anionic surfactant or a nonionic surfactant. Said substrate has a concavo-convex portion made of concave and convex portions on its surface, a substrate on the upper end face is selective binding substance immobilized in the convex portion, the chip for analysis according to claim 1 or 2 . It said cover member has one or more through holes communicating with the void, analyzing chip according to any one of claims 1-3. The shortest distance interval between the selective binding substance immobilized surface and the cover member of the substrate, fine particles is less than the diameter, the analysis chip according to any one of claims 1-4. The analytical chip according to any one of claims 1 to 5 , wherein the selective binding substance is DNA, RNA, protein, peptide, sugar, sugar chain or lipid. A substrate having a selective binding substance immobilized on the surface thereof and a cover member bonded to the substrate, and having a gap between the substrate and the cover member, the fine particles can be movably stored in the gap. is, the analysis includes an analysis chip for surface active agent is coated on the surface of the fine particles, the reaction solution containing the nonionic surfactant a reaction solution for binding reaction of the selective binding substance and the analyte For kit.

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