WO2002004951A1 - Plaque de micro-titration - Google Patents

Plaque de micro-titration Download PDF

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Publication number
WO2002004951A1
WO2002004951A1 PCT/JP2001/002821 JP0102821W WO0204951A1 WO 2002004951 A1 WO2002004951 A1 WO 2002004951A1 JP 0102821 W JP0102821 W JP 0102821W WO 0204951 A1 WO0204951 A1 WO 0204951A1
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WIPO (PCT)
Prior art keywords
microarray
carrier
frame
molecule
sample
Prior art date
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PCT/JP2001/002821
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English (en)
Japanese (ja)
Inventor
Akio Yamane
Toru Miyagi
Original Assignee
Wakunaga Pharmaceutical Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wakunaga Pharmaceutical Co., Ltd. filed Critical Wakunaga Pharmaceutical Co., Ltd.
Priority to AU2001244687A priority Critical patent/AU2001244687A1/en
Publication of WO2002004951A1 publication Critical patent/WO2002004951A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention relates to a microarray used for analyzing biomolecules, and more particularly, to a diagnostic microarray suitable for processing a large amount of a sample.
  • Analysis of gene expression at the mRNA level can be performed by analyzing a specific known gene sequence for a sample, as in the Northern blot method (Alwine JC et al. Proc Natl Acad Sci USA. 1977 Dec; 74 (12): 5350-4). There is a method used as a probe. On the other hand, conversely, genes with different expression levels among different samples, such as the differential display method (Liang P and Pardee AB. Science. 1992 Aug 14; 257 (5072): 967-71), are used. There are ways to find them.
  • Northern blotting is time-consuming and labor-intensive, and the number of genes that can be analyzed is limited. Although it is a reliable and convenient method for analyzing a small number of candidate genes, it is very difficult to analyze the vast number of genes identified one after another.
  • the differential display method a sample extracted from cells and tissues in different states and a sample amplified at random are electrophoresed, and band patterns are compared to extract bands with different intensities between different samples. And determine the base sequence. In many cases, this band does not contain the entire nucleotide sequence of the gene, and the gene is identified by comparing the partial sequence obtained from the band with the previously determined gene sequence data. You. The function can be estimated from the state of the identified gene that was strongly expressed in the cell.
  • a technique called DNA microarray or DNA chip is capable of analyzing a much larger number of genes than these methods.
  • a DNA microarray is a solid plate in which a partial amplification product of a known gene sequence or an oligonucleotide is immobilized on a small plate such as a slide glass at a high density. Thousands to tens of thousands of types are obtained by hybridization. This technology allows simultaneous detection of gene expression levels on a single slide.
  • DNA can be directly solid-phase synthesized on a carrier, or can be immobilized on a carrier by spotting a previously synthesized DNA with a fine pin or the like.
  • DNA microarrays are being developed as a very powerful technique in expression analysis and polymorphism analysis research, and clinical applications are expected to become more active in the future based on these research results ( Hodgson J. Nat Biotechnol. 1998 Aug; 16 (8): 725-7).
  • the probe is placed on a slide glass.
  • the sample solution is spread over the entire probe area by immobilizing a solid phase, placing a hybridization solution containing a detection target on a probe, and covering the probe with a cover slip.
  • the target nucleic acid group is labeled with a fluorescent substance or the like, and after hybridization, excess labeled nucleic acid not hybridized to the probe is removed by washing.
  • This method has a problem that bubbles easily enter the sample solution when a force bar slip is applied.
  • the mixed bubbles inhibit contact between the probe and the sample and cause false negatives.
  • very careful handling of force bar slips is required, and the processing of large samples takes a great deal of time.
  • the hybridization solution is present in a narrow space covered with a cover slip, and is difficult to disperse, resulting in a bias in the sample concentration, causing false negatives due to insufficient sample volume and false positives due to excessive sample volume.
  • the hybridization solution In order for the hybridization solution to be present in the force bar slip region without excess or deficiency, it is necessary to finely adjust the amount of the solution.
  • the hybrid solution When two or more probe regions are present on the same carrier and different samples are allowed to react with each probe region on this carrier, if the hybrid solution is excessive, it will be outside the cover slip region, that is, outside the probe region.
  • the hybridization solution protrudes, causing the contamination of the sample with other probe regions, which causes false positives due to cross-hybridization.
  • the force purse slip after finishing the hybridization, the force purse slip must be removed for cleaning, but the cover slip is strongly adsorbed on the carrier due to surface tension, and it is difficult to quickly remove it.
  • the present inventors improve the operability of hybridization and the reliability of analysis data by providing a frame in the detection part of the microarray and holding the sample (test liquid) on the detection part. was found.
  • An object of the present invention is to provide a microarray excellent in operability and reliability of analysis data and suitable for processing a large number of samples.
  • a microarray for detecting a plurality of target molecules a carrier for detecting a target molecule to which a molecule capable of capturing the target molecule (capture molecule) may be immobilized,
  • a microarray having at least one unit comprising a frame for holding a sample on the carrier is provided.
  • the microarray according to the present invention does not have an immobilized lid on the frame, the captured molecule can be immobilized at any time. This makes it easy to discharge and inject the washing liquid.
  • the frame is integrated in the microarray according to the present invention in advance, the work of attaching the frame in the detection step is unnecessary, and the work of removing the frame is not required because the frame does not hinder detection of the target molecule. Therefore, the microarray according to the present invention is advantageous in that it has excellent operability.
  • the microarray according to the present invention can stably hold a solution on the detection portion, it is possible to use a probe that can be detected only in a solution such as a molecular beacon probe having excellent reliability and immediacy. Become. Further, in the microarray according to the present invention, even when a plurality of samples are handled on the same carrier, contamination of each sample into another sample can be avoided by clearly dividing a plurality of detection regions by a frame. Therefore, the microarray according to the present invention is advantageous in that a reliable data without erroneous determination can be obtained and a large amount of sample can be processed. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a diagram showing a microarray according to the present invention.
  • Azusa 3 is attached to carrier polystyrene 2.
  • a region surrounded by a frame 3 on the carrier polystyrene 2 is a hybridization region.
  • A is a plan view
  • B is a sectional view
  • C is a perspective view.
  • FIG. 2 is a diagram showing a microarray according to the present invention.
  • a plurality of frames 3 are detachably attached to the carrier polystyrene 2.
  • a region surrounded by a frame 3 on the carrier polystyrene 2 is a hybridization region.
  • A is a plan view
  • B is a sectional view
  • C is a perspective view.
  • FIG. 3 is a diagram showing a microarray according to the present invention.
  • a plurality of frames 3 integrated by the frame holders 14 are detachably attached to the carrier polystyrene 2 c.
  • a region surrounded by the frame 3 on the carrier polystyrene 2 is a hybridization region.
  • FIG. 4 shows a microarray set.
  • A is a diagram showing a microarray 5 and a microarray holder 16.
  • B is a diagram showing a microarray set 7 obtained by placing a plurality of microarrays 5 in a box-shaped microarray holder 6.
  • FIG. 5 is a diagram showing an embodiment in which a plurality of microarrays are integrated by fitting concave portions 8 and convex portions 9 provided on the carrier 2 and the frame holder 4 to each other.
  • a microarray set can be obtained by integrating a plurality of microarrays at uneven portions.
  • FIG. 6 is a view showing an embodiment in which a plurality of microarrays are integrated by a clip-shaped microarray holder 10.
  • a microarray set can be obtained by integrating a plurality of microarrays with the clip 10.
  • FIG. 7 is a diagram showing a frame according to the present invention.
  • a plurality of frames 3 are integrated by a frame holder 4.
  • FIG. 8 is a diagram showing the results of HLADRB1 gene tying in the examples. Detailed description of the invention
  • microarray refers to a carrier in which a group of arranged capture molecules is immobilized on a carrier or a carrier capable of immobilizing the same, and is referred to as a “detection chip” such as a DNA chip or the like.
  • the microarray according to the present invention which is used to include a carrier for preparation, comprises a carrier for detecting a target molecule and a sample (test liquid) for detecting the presence of the target molecule on a detection portion. And at least one unit consisting of: It preferably has 2 to 10 units, more preferably 2 to 5 units.
  • unit means a detection space separated by a frame.
  • the carrier constituting each unit can be a common carrier. That is, a plurality of units can be formed by installing a plurality of frames on a common carrier. See, for example, FIGS.
  • the capture molecule may be immobilized on the carrier in advance.
  • Target molecules to be detected include antigenic substances (proteins, glycoproteins, amino acids, etc.) or antibodies against them, nucleic acids (DNA, RNA, etc.), peptide nucleic acids, etc., especially during organ or bone marrow transplantation.
  • antigenic substances proteins, glycoproteins, amino acids, etc.
  • nucleic acids DNA, RNA, etc.
  • peptide nucleic acids etc., especially during organ or bone marrow transplantation.
  • HLA white blood cell type It is considered useful for tying.
  • the capture molecules for detecting these are antibodies against substances having antigenicity or substances having antigenicity, nucleic acid probes, nucleic acid probes (Hermann T and Patel DJ. Science. 2000 Feb 4; 287 (5454): 820 -5) or a peptide nucleic acid (Beier M and Hoheisel JD.Nucleic Acids Res. 1999 May l; 27 (9): 1970-7), etc., and a molecule such as streptavidin that has the ability to bind to the molecule to be detected. It may be. Preferably, it is a nucleic acid probe.
  • nucleic acid probe a synthetic oligonucleotide, a plasmid / phage, a nucleic acid amplified in a living body such as ssMA, or a nucleic acid fragment amplified in a test tube by PCR or the like can be used.
  • the capture molecules can be immobilized on the carrier by physical adsorption or chemical bonding.
  • the carrier glass surface was coated with poly-L-Lys and the positive charge of the Lys amino group was observed.
  • a typical example is one utilizing the electrostatic action between the negative charge of the phosphate bond between DNA and DNA.
  • the microphone mouth plate Inouye S and Hondo R. J Clin Microbiol. 1990
  • the capture molecules are preferably arranged on a carrier and immobilized at a high density.
  • the amount of information obtained in a single experiment is dramatically increased due to the high density of the probe, and changes in the state of the living body caused by environmental changes, diseases, administration of drugs, etc. It can be captured as an image.
  • Probe densification is described, for example, in Lipshutz RJ et al. Nat Genet. 1999 Jan; 21 (l Suppl): 20-4 and Schena M et al. Science. 1995 Oct 20; 270 (5235): 467-70.
  • the frame constituting the unit has a height sufficient to hold the test liquid on the detection portion, preferably 3 mm to 15 mm, more preferably 5 mm to 1 O mm It is.
  • the microarray according to the present invention is provided with a frame. If the frame and the carrier are integrated in advance, there is no need to attach the frame before detection. In addition, the detection of the target molecule is not hindered by the frame, so that the removal operation is not required. Therefore, it is advantageous in that the operation procedure can be simplified.
  • the frame can be removable if required.
  • the frame and the carrier can be reversibly bonded with a silicon gasket or the like.
  • the frame may have one or more projections or depressions
  • the carrier may have depressions or projections corresponding to the projections or depressions of the frame.
  • the frame and the carrier can be detachably fixed by fitting the projection or the recess of the frame with the corresponding recess or the projection of the carrier.
  • a plurality of units may be integrated by a frame holder.
  • a plurality of units may be integrated in the opening of the frame.
  • Such an embodiment is as shown in FIG.
  • a plurality of units may be integrated on one carrier so as to be arranged in a line.
  • the shape of the unit is preferably a rectangular parallelepiped, a cube, or a similar shape suitable for immobilizing the captured molecules efficiently and at high density.
  • the purpose is to prevent the test liquid from scattering and evaporating to the unit as necessary.
  • the bottom surface of a conventional microplate is a circle, and when a plurality of independent regions are arranged in parallel, capture molecules are immobilized between the regions. There were regions where the capture molecules could not be created, and the capture molecules could not be arranged efficiently.
  • the aligned capture molecules can be immobilized on the carrier at high density, and the immobilized portion can be efficiently arranged, so that one microarray can process many samples at a time. This is advantageous in that it can be performed.
  • a carrier portion where a sample solution is to be present (for example, a probe-immobilized region) is surrounded by a frame, and this frame is moved out of the probe region of the sample solution (for example, a hybridization solution). Block oozing. Therefore, the sample solution can be injected into the carrier portion without worrying that the sample solution spreads to other regions, which is advantageous in that the operation is simplified.
  • the sample solution is freely stirred by shaking.
  • the concentration of the target molecule in the solution is uneven and the bubbles from remaining on the capture molecule. Accordingly, it is advantageous in that more stable hybridization can be performed. Since the frame of the microarray according to the present invention is not covered with the lid on which the frame is fixed, the capture molecules can be immobilized at any time. Therefore, the operation procedure can be made flexible.
  • the detection area is set using an aspire with a nozzle and a dispenser. Can be washed.
  • the aspirator and the dispenser can be, for example, a microplate washer. Therefore, the microarray according to the present invention is advantageous in that the washing can be automated as in the microplate washing. Note that it is also possible to automate the washing process when preparing a microarray.
  • the removal of the washing solution after the hybridization was performed by centrifugation or air injection pressure.
  • the salt of the washing solution after the hybridization often remains. Since the salt remaining on the surface itself fluoresces, it caused non-uniform noise when the scanner sensitivity was increased, hindering the detection of trace samples. In addition, the salt remaining on the probe spot became a large crystal, which sometimes caused a false negative by blocking the excitation light and the fluorescence.
  • the present invention is also advantageous in that detection can be performed while the detection solution is retained, and such a decrease in detection sensitivity is avoided.
  • a plurality of hybridization regions can be formed by arranging frames as necessary.
  • the fact that a plurality of nodipase regions are physically separated by a frame indicates that highly homologous genes such as the human leukocyte antigen genes HLA-A and HLA-B are detected on the same carrier.
  • HLA-A and HLA-B highly homologous genes
  • it is very effective to avoid cross-hybridization caused by contamination of each gene amplification product into the other hybridization region. Detecting multiple genes from the same sample on the same microarray avoids erroneous determination due to a combination error of each gene sequence information that can occur due to a notation error on the container, etc., compared to using multiple containers. This is advantageous in that it can be performed.
  • the microarray of the present invention can stably hold a solution on a probe, the function can be exhibited only in a solution such as a molecular beacon probe if a device capable of measuring with a frame is used. Inappropriate props can be used. In diagnosis, high reliability and immediacy are required, and it is important to prevent false negatives due to contamination of nucleic acid amplification products with reagents before the gene amplification reaction and to shorten the detection time. Ishi guro T et al. Nucleic Acids Res. 1996 Dec 15; 24 (24): 4992-7, as shown in Orizu E et al. Molecular & Cellular Probes.
  • hybrid recognition probes such as the molecular beacon shown in 1998 Aug; 12 (4): 219-26
  • hybrid recognition probes do not require sample labeling and do not require further washing. Since detection is possible, the detection system is very simple and the risk of contamination is low. Therefore, the hybrid recognition probe is expected to be applied in the diagnostic field.
  • Application as a microarray probe has been attempted, but molecular beacons can be detected in a dry state as pointed out in Steemers FJ. Et al. Nature Biotechnology. 2000 Jan; 18 (l): 91-4.
  • it is difficult to dry with conventional microarrays and molecular beacons cannot be used as microarray probes because drying is indispensable for scanning.
  • microarray container according to the present invention is advantageous in that a hybrid recognition probe excellent in reliability and immediacy can be used since detection can be performed while a liquid is retained on a prop.
  • a removable lid can be used if necessary.
  • the lid prevents evaporation of the hybridization solution in the hybridization.
  • Evaporation of the hybridization solution is a problem because it changes the salt concentration of the hybridization solution, making it impossible to obtain the predetermined hybridization conditions.
  • the scattering of the hybridization solution containing the nucleic acid amplification product leads to serious inspection errors when it contaminates the amplification system. Therefore, the lid can be easily removed while maintaining the required hermeticity for operational reasons during the immobilization of the probe, injection of the sample onto the carrier surface during hybridization, and the washing process after hybridization. desirable.
  • the required tightness is sufficient to avoid evaporation that can cause large fluctuations in the liquid volume during hybridization and does not have to be strong enough to prevent the liquid from leaking out. Therefore, the lid can be a simple seal.
  • a lid or frame can be provided with an inlet or outlet for the hybridization solution to automate a series of reactions.
  • the carrier portion, frame, and, in some cases, the lid for detecting the target molecule according to the present invention are not particularly limited as long as they have a material having an impermeable surface, but when the detection is performed from the bottom of the microarray, it is preferably transparent.
  • Materials can be used. Examples of transparent materials include glass, plastics such as polystyrene and polypropylene, and processed products obtained by chemical treatment of these materials as required, and processed products coated with polymers such as nitrocellulose and polyacrylamide. .
  • the detection sensitivity can be increased by coating with aluminum or coloring the material black.
  • ultraviolet rays Zouali M and Stollar BD. J Immunol Methods.
  • the thickness of the microarray is not particularly limited, but is preferably such that it does not affect the light transmittance, and is generally about 0.5 mm to 2 mm.
  • a carrier for detecting a target molecule to which a molecule capable of capturing the target molecule (capture molecule) may be immobilized, and a frame for holding the sample on the carrier are provided.
  • Target molecule having multiple units consisting of Provided is a microarray for detecting the number of units, wherein a plurality of units are integrated by a frame holder, and a plurality of units are formed on a common carrier.
  • a carrier for detecting a target molecule to which a molecule capable of capturing the target molecule (capture molecule) may be immobilized, and a frame for holding a sample on the carrier are provided.
  • a microarray for detecting a target molecule comprising 2 to 10 units consisting of: a unit formed on the same carrier, and a unit formed by a frame holder at an opening of the frame.
  • An integrated microarray is provided.
  • a carrier for detecting a molecule of interest to which a molecule capable of capturing the molecule of interest (capture molecule) may be immobilized, and a sample held on a carrier
  • a microarray for detecting a target molecule comprising 2 to 10 units comprising a frame, a plurality of units being formed on a common carrier, and a plurality of units being formed at an opening of the frame.
  • a microarray is provided in which the frame is detachably fixed to the carrier by fitting the convex or concave portion of the frame with the concave or convex portion of the corresponding carrier.
  • a microarray set in which a plurality of microarrays according to the present invention are integrated.
  • a plurality of microarrays can be integrated by a microarray holder.
  • the microarray holder may be any one that can align and fix a plurality of microarrays, and may be, for example, a box-shaped holder (see Fig. 4) or a clip-shaped holder (see Fig. 6).
  • a plurality of microarrays can also be integrated by fitting one or more protrusions and / or recesses provided on the microarray (see FIG. 5). The uneven portion may be present on both the carrier and the frame holder, or may be present on either of them.
  • the microarray set integrated by the uneven portion may be further supported by a microarray holder (for example, a box-shaped holder).
  • the number of microarrays included in the microarray set is preferably 2 to 12.
  • a frame for holding a sample on a carrier for detecting a target molecule of a microarray wherein the frame is used by being immobilized on the carrier.
  • a frame is provided. This frame may be detachable from the carrier part.
  • This frame may also be provided with one or more convex portions or concave portions corresponding to one or more concave portions or convex portions provided on the carrier.
  • the frame can be detachably fixed to the carrier by fitting the convex or concave portion of the frame with the corresponding concave or convex portion of the carrier.
  • the sample can be a PCR amplified nucleic acid sample.
  • a nucleic acid label a fluorescent substance such as Cy3, a radioisotope, or a substance having an affinity for another substance such as hapten-dibiotin such as DNP can be used.
  • These labels can be prepared by using a primer whose terminal is labeled with an arbitrary labeling substance, or by introducing the labeling substance into a monomeric nucleic acid used for amplification in advance.
  • DNP or biotin it is necessary to add a substance (enzyme-labeled antibody or fluorescently-labeled streptavidin) that enables detection of these chemicals at the time of detection.
  • the probe is immobilized on the microarray according to the present invention
  • the probe is immobilized on the detection portion.
  • the sample is poured into the microarray according to the present invention, and the target nucleic acid is hybridized with the probe. Remove and wash the sample if necessary.
  • the target nucleic acid is detected. Even when detection is performed by chemiluminescence or color development, a stable result can be obtained because the reaction solution is stably maintained on the probe.
  • ELSA Endoza LG et al. Biotechniques. 1999 0ct; 27 (4 ): 778-80, 782-6, 788. It can also be used to analyze interactions between proteins (Emili AQ and Cagney G. Nat Biotechnol. 2000 Apr; 18 (4): 393-7).
  • a microarray container was prepared in which a frame was attached on a flat polystyrene plate so that the hybridization area was 152 mm 2 (19 mm ⁇ 8 mm).
  • 15 types of human leukocyte antigen gene DRB 1 typing probes (# 1: DRB 2801 J, # 2: DRB1002J, # 3: DRB7004, # 4: DRB 1004, # 5: DRB5703J, # 6DRB3705J, # 7: DRB 10X YJ , # 8: DRB 1006, # 9: DRB70J, # 10: DRB 1007, # 11: DRB 1008J, # 12: DRB 1003NJ, # 13: DRB 1005, # 14: DRB7003, # 15: c ommon It was synthesized according to the method described in Kawai S et al. Eur JI employed unogenet. 1996 Dec; 23 (6): 471-86. This probe was spotted by a frame on the area formed on the polystyrene
  • the partial sequence of allyl * 1101 of the DRB1 gene was amplified by PCR according to the method described in Kawai S et al. Eur J I uno genet. 1996 Dec; 23 (6): 471-86. However, the 5 'end of the primer was labeled with fluorescein instead of the biotin described in the literature to obtain a fluorescein-labeled amplification product.
  • the PCR product was purified, diluted with 1 OxSSC to a final concentration of 1 OnM, and used as a solution for hybridization.
  • the hybridization solution was removed, and the plate was washed three times with 1 ⁇ SSC.
  • the container according to the present invention enables gene detection and typing. It became clear.

Abstract

Cette invention concerne une plaque de micro-titration qui est remarquable en termes d'exploitabilité et de fiabilité des données, et qui convient pour le traitement à fort rendement de spécimens. Cette plaque de micro-titration pour détection d'une molécule cible, comprend au moins une unité composée d'un support pour la détection d'une molécule cible permettant de fixer une molécule-cible/molécule capturable (molécule de capture), et un cadre permettant de retenir un échantillon sur le support.
PCT/JP2001/002821 2000-07-10 2001-03-30 Plaque de micro-titration WO2002004951A1 (fr)

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AU2001244687A AU2001244687A1 (en) 2000-07-10 2001-03-30 Micro-array

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JP2000-208317 2000-07-10
JP2000208317 2000-07-10

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003227841A (ja) * 2002-02-01 2003-08-15 Fuji Photo Film Co Ltd リセプター・リガンド会合反応方法
JP2004045179A (ja) * 2002-07-11 2004-02-12 Nisshinbo Ind Inc マイクロアレイ用基材
JP2004138571A (ja) * 2002-10-21 2004-05-13 Joji Inasawa 生化学的検出マイクロアレイを用いる効率的検出用機器
JP2004177345A (ja) * 2002-11-28 2004-06-24 Sumitomo Bakelite Co Ltd マイクロアレイ用プラスチック基板
JP2005515773A (ja) * 2002-01-28 2005-06-02 ナノスフェアー　インコーポレイテッド ハイブリダイゼーションの装置および方法
JP2005185171A (ja) * 2003-12-25 2005-07-14 Canon Inc Ｈｌａ−ｄｒアレルを同定するためのプローブセット及び特定方法
JP2006509201A (ja) * 2002-12-04 2006-03-16 インターナショナル・ビジネス・マシーンズ・コーポレーション 表面処理方法
US7264845B2 (en) 2003-04-29 2007-09-04 The Procter & Gamble Company Method for increasing the hydrophobicity of a lavatory bowl surface
US9863884B2 (en) 2012-08-10 2018-01-09 Hamamatsu Photonics K.K. Surface-enhanced Raman scattering element, and method for producing same
US9863883B2 (en) 2012-08-10 2018-01-09 Hamamatsu Photonics K.K. Surface-enhanced raman scattering element
US10408761B2 (en) 2012-08-10 2019-09-10 Hamamatsu Photonics K.K. Surface-enhanced Raman scattering element
JP2020016658A (ja) * 2012-09-28 2020-01-30 ヴィブラント ホールディングス リミテッド ライアビリティ カンパニー 生体分子解析のための方法、システム、およびアレイ
US11565231B2 (en) 2012-02-07 2023-01-31 Vibrant Holdings, Llc Substrates, peptide arrays, and methods
US11674956B2 (en) 2012-09-28 2023-06-13 Vibrant Holdings, Llc Methods, systems, and arrays for biomolecular analysis

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JPS6025962U (ja) * 1983-07-27 1985-02-21 山本　康裕 検尿紙
JPH01102360A (ja) * 1987-10-16 1989-04-20 Nissha Printing Co Ltd 反応試験器
JPH01102869U (fr) * 1987-12-28 1989-07-11

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6025962U (ja) * 1983-07-27 1985-02-21 山本　康裕 検尿紙
JPH01102360A (ja) * 1987-10-16 1989-04-20 Nissha Printing Co Ltd 反応試験器
JPH01102869U (fr) * 1987-12-28 1989-07-11

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005515773A (ja) * 2002-01-28 2005-06-02 ナノスフェアー　インコーポレイテッド ハイブリダイゼーションの装置および方法
JP2003227841A (ja) * 2002-02-01 2003-08-15 Fuji Photo Film Co Ltd リセプター・リガンド会合反応方法
JP2004045179A (ja) * 2002-07-11 2004-02-12 Nisshinbo Ind Inc マイクロアレイ用基材
JP2004138571A (ja) * 2002-10-21 2004-05-13 Joji Inasawa 生化学的検出マイクロアレイを用いる効率的検出用機器
JP2004177345A (ja) * 2002-11-28 2004-06-24 Sumitomo Bakelite Co Ltd マイクロアレイ用プラスチック基板
JP2006509201A (ja) * 2002-12-04 2006-03-16 インターナショナル・ビジネス・マシーンズ・コーポレーション 表面処理方法
US7264845B2 (en) 2003-04-29 2007-09-04 The Procter & Gamble Company Method for increasing the hydrophobicity of a lavatory bowl surface
JP2005185171A (ja) * 2003-12-25 2005-07-14 Canon Inc Ｈｌａ−ｄｒアレルを同定するためのプローブセット及び特定方法
US11565231B2 (en) 2012-02-07 2023-01-31 Vibrant Holdings, Llc Substrates, peptide arrays, and methods
US9863884B2 (en) 2012-08-10 2018-01-09 Hamamatsu Photonics K.K. Surface-enhanced Raman scattering element, and method for producing same
US9863883B2 (en) 2012-08-10 2018-01-09 Hamamatsu Photonics K.K. Surface-enhanced raman scattering element
US10408761B2 (en) 2012-08-10 2019-09-10 Hamamatsu Photonics K.K. Surface-enhanced Raman scattering element
JP2020016658A (ja) * 2012-09-28 2020-01-30 ヴィブラント ホールディングス リミテッド ライアビリティ カンパニー 生体分子解析のための方法、システム、およびアレイ
US11674956B2 (en) 2012-09-28 2023-06-13 Vibrant Holdings, Llc Methods, systems, and arrays for biomolecular analysis
US11815512B2 (en) 2012-09-28 2023-11-14 Vibrant Holdings, Llc Methods, systems, and arrays for biomolecular analysis

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