Methods for analyzing specimens and specimen analyzers

Abstract

To solve the problem that analysis using an array plate usually requires a drying step that interrupts a series of tasks, and non-uniform drying of the array plate has been one of the causes of reduction in reproducibility.SOLUTION: A method proposed herein involves acquiring optical information of a plurality of spots while the spots are in contact with an observation liquid.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a specimen analysis apparatus for analyzing a specimen and a method for analyzing a specimen.

Background Art

[0002] Array plates such as protein arrays, peptide arrays, and DNA arrays in which a large number of substances such as proteins, peptides, and nucleic acids are spot-fixed on a substrate are known. By using an array plate, the interaction between a large number of fixed substances and the substances in a specimen can be observed at once. Therefore, the interactions with a large number of substances including biological specimens such as blood, cell extracts, saliva, and interstitial fluid are comprehensively analyzed.

[0003] As a measurement method using an array plate, a method is known in which spots where an interaction of interest has occurred are selectively fluorescently labeled to obtain optical information. In Patent Documents 1 and 2, an array plate is observed using a microarray scanner. The apparatus of Patent Document 1 has an irradiation optical system, a fluorescence detection optical system, and a two-dimensional scanning system. The irradiation optical system has a function of condensing and irradiating a laser beam onto the array plate. The fluorescence detection optical system has a function of detecting the amount of fluorescence from the fluorescently labeled spots. The two-dimensional scanning system has a function of obtaining a fluorescence image of the spots on the array plate by two-dimensionally scanning the array plate or the optical system.

[0004] Patent Document 2 proposes an antibody array using proximity mediation for detecting an oncogenic fusion protein.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

[0006] However, Patent Documents 1 and 2 have the problem that the array plate must be observed in a dry state. The process of bringing an array plate into contact with a sample and selectively fluorescently labeling the spots where the reaction of interest occurs is usually performed in liquid. Therefore, the drying process interrupts this process. Conventional drying processes have used physical removal methods such as centrifugation and air blowing to physically remove the liquid in contact with the plate, rather than methods that include a heating process utilizing the phase transition between the liquid and gas phases, in order to reduce the denaturation of biological materials and samples. In this specification, from this point forward, the drying process will be used as a concept that includes a process of removing liquid from the substrate using external forces, including inertial forces, which do not necessarily require a heating process. The purpose of the drying process is often to remove residual components from the plate that are not fixed to spots among the dissolved components in reagents such as targets, enzymes, and antibodies, i.e., components that are not needed in subsequent processes. Therefore, it is not intended to selectively volatilize the solvent and leave the dissolved components as residue on the substrate.

[0007] On the other hand, when a drying process was performed on the array plate in contact with the reagent, the inventors of the present invention visually confirmed that, as shown in Figure 18, traces of some of the reagent's liquid remained even after drying, resulting in "uneven drying." It was presumed that the liquid traces were areas where reagents containing a higher concentration of dissolved components were present locally for a longer period than in other areas outside the liquid traces, and therefore it was presumed that more residue was present in the liquid traces than in other areas. In other words, from the reaction process to the analysis process, it is desirable that multiple spots on the array plate undergo a uniform history. However, the drying process was one of the factors that limited the uniformity and reproducibility of this history. In Figure 18, 199 represents the array plate after drying, 193-1 to 193-36 represent spots on the array plate, and 190 represents a liquid trace. Liquid trace 190 indicates that the drying of the reaction solution was slower compared to the surrounding area where liquid trace 190 is not observed. This raised concerns that some of the dissolved components in sample 143, phosphorylation reaction solution, and reagents remained as localized residues compared to areas other than those with liquid trace 190. Although such liquid traces 190 are visible traces, the detection dynamic range when optically detecting information from labels is about 2 to 5 orders of magnitude. Therefore, there were concerns that a sample introduction process that resulted in uneven drying would become a source of background noise in the analysis process.

[0008] Therefore, the present invention aims to provide an analytical method and analytical apparatus that reduce the influence of residuals due to uneven drying in the analysis of samples using array plates. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide an analytical method and analytical apparatus that reduce the influence of residuals due to uneven drying when analyzing a sample using an array plate. [Brief explanation of the drawing]

[0010] [Figure 1] This is a diagram illustrating the first embodiment (a) and the second embodiment (b). [Figure 2] This diagram illustrates the first labeling process and the second labeling process. [Figure 3] This figure shows the apparatus according to this embodiment. [Figure 4] This figure shows the schematic configuration (a) and desktop arrangement (b) of the sample analyzer according to this embodiment. [Figure 5] This is a diagram illustrating the process of Example 1. [Figure 6]It is a diagram for explaining the process of Example 1. [Figure 7] It is a diagram for explaining the process of Example 1. [Figure 8] It is a diagram for explaining the process of Example 1. [Figure 9] It is a diagram for explaining the fluorescence detection step of Example 1. [Figure 10] It is a diagram for explaining the structure of the frame. [Figure 11] It is a diagram for explaining the optical information acquisition method. [Figure 12] It is a diagram for explaining the array plate support base. [Figure 13] It is a diagram for explaining the reagent liquid discharge method using the array plate tilting mechanism. [Figure 14] It is a diagram for explaining the second array plate tilting mechanism. [Figure 15] It is a diagram for explaining the third array plate tilting mechanism. [Figure 16] It is a diagram for explaining the structure of the second frame. [Figure 17] Shows the analysis results. [Figure 18] It is a diagram showing the liquid traces on the array plate.

Mode for Carrying Out the Invention

[0011] The analysis method according to an embodiment of the present invention is an analysis method for analyzing a specimen containing a target using an array plate having a plurality of spots containing a biological substance on one surface, a specimen introduction step of bringing the specimen into contact with the plurality of spots and reacting a part of the plurality of spots with the target, a specimen amount reduction step of reducing the amount of the specimen in contact with the plurality of spots, an observation liquid introduction step of bringing the surface into contact with the observation liquid so that the plurality of spots come into contact with the observation liquid, and an acquisition step of acquiring optical information of the plurality of spots in a state where the plurality of spots are in contact with the observation liquid. A first labeling step of labeling the target before the sample introduction step, and The analytical method further includes at least one of the following: a second labeling step performed after the sample volume reduction step, which labels the spot that has reacted with the target.

[0012] Furthermore, an analytical apparatus according to another embodiment of the present invention is A mounting section on which an array plate having multiple spots containing biological material on one side is placed, A holding portion that holds the liquid so that it is in contact with one of the aforementioned surfaces, A liquid supply / drainage unit that enables the introduction of the liquid into the liquid-holding region of the holding unit, or the discharge of the liquid held by the holding unit, A moving part that moves the liquid supply and drainage part relative to the previously described mounting part, An optical system that irradiates light onto the plurality of spots and detects the light from the plurality of spots, A scanning unit that moves the optical system relative to the previously described mounting unit, The analyzer comprises a control unit that controls the timing for executing each of the following steps: a sample introduction step of bringing a sample into contact with the plurality of spots and reacting a portion of the plurality of spots with a target contained in the sample; a sample volume reduction step of reducing the amount of the sample in contact with the plurality of spots; an observation solution introduction step of bringing the surface into contact with the observation solution so that the plurality of spots are in contact with the observation solution; and an acquisition step of acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, wherein the control unit controls the timing for executing at least one of the following steps: a first labeling step performed before the sample introduction step to label the target, and a second labeling step performed after the sample volume reduction step to label the spots that have reacted with the target.

[0013] Furthermore, an analytical apparatus according to another embodiment of the present invention A mounting section on which an array plate having multiple spots containing biological material on one side is placed, A holding portion that holds the liquid so that it is in contact with one of the aforementioned surfaces, A liquid supply / drainage unit that enables the introduction of the liquid into the liquid-holding region of the holding unit, or the discharge of the liquid held by the holding unit, A moving part that moves the liquid supply and drainage part relative to the previously described mounting part, An optical system that irradiates light onto the plurality of spots and detects the light from the plurality of spots, The optical system has a scanning unit that moves it relative to the previously described mounting unit, An analytical apparatus comprising: a sample introduction step of bringing a sample into contact with the plurality of spots and reacting a portion of the plurality of spots with a target contained in the sample; a sample volume reduction step of reducing the amount of the sample in contact with the plurality of spots; an observation solution introduction step of bringing the surface into contact with the observation solution so that the plurality of spots are in contact with the observation solution; and an acquisition step of acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution; and further comprising at least one of a first labeling step of labeling the target before the sample introduction step, and a second labeling step performed after the sample volume reduction step of labeling the spots that have reacted with the target, the apparatus comprising a control unit that controls the above-described placement unit, a liquid supply and drainage unit, the moving unit, the optical system, and the scanning unit, so as to be able to perform each step of an analytical method.

[0014] As one embodiment, the present invention provides an analytical method for analyzing a sample containing a target using an array plate having a plurality of spots containing a biological substance on one surface, comprising: a sample introduction step of bringing the sample into contact with the plurality of spots and reacting a portion of the plurality of spots with the target; a sample volume reduction step of reducing the amount of the sample in contact with the plurality of spots; an observation solution introduction step of bringing the surface into contact with the observation solution so that the plurality of spots are in contact with the observation solution; and an acquisition step of acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, further comprising at least one of a first labeling step of labeling the target before the sample introduction step, and a second labeling step performed after the sample volume reduction step of labeling the spots that have reacted with the target.

[0015] <Array Plate> An array plate has spots containing various types of biological materials on a substrate and is used for comprehensive analysis of samples. Array plates are sometimes called microchips, microarrays, protein chips, DNA chips, etc. The location of each spot on an array plate is called its address. Each spot can be assigned a symbol as its address. The address may also be determined by the physical position of each spot on the array plate. In that case, it can be determined by the distance from each edge of the array plate or by coordinates.

[0016] In this specification, an array plate has at least 16 spots containing biomaterial, and includes at least two spots in which the composition, content, or blending amount of the biomaterial differs from each other. In this specification, an array plate is flat and has spots containing biomaterial on one side. The side on which the spots are provided may be referred to as the first side (101-1). The back surface of the first side may be referred to as the other side (101-2) or the second side (101-2).

[0017] The array plate in this invention is a flat plate with a length and width of 10 mm to 128 mm and a thickness of 0.1 mm to 2.5 mm. The shape of the plate can be a cuboid, a hexahedron including a truncated square pyramid, a cylinder, an elliptical cylinder, or a disc shape including a truncated cone. The periphery defining the edges of the plate faces may preferably be rounded or tapered. Multiple spots are arranged on one of two opposing faces that define the thickness of the plate. The diameter of the spots is adjusted according to the size of the plate, the imaging field, and the number of spots, but is typically between 0.1 μm and 5000 μm. The spots are spaced apart from adjacent spots and can be square, rectangular, circular, or elliptical. The arrangement period between spots is typically between 1.1 and 10 times the diameter of the spots. One array plate has at least 16, more preferably 64, and even more preferably 1000 or more spots.

[0018] Commercially available array plates can be used. Microarray plates are sold by Agilent Technologies, Inc., RayBiotech, and others. Alternatively, array plates can be prepared by referring to known methods. Array plates are prepared by immobilizing the desired biomaterial on one surface of a suitable substrate. Immobilization can also be referred to as adsorption, and includes immobilization by hydrophobic interactions, electrostatic interactions, van der Waals interactions, hydrogen bonds, and covalent bonds. The substrate is transparent to the wavelength of excitation light. Depending on the optical arrangement, the substrate may also be transparent to the wavelength of fluorescence. Translucency means the property of transmitting light of at least one wavelength. Translucency includes the transmission of a portion of light of a specific wavelength. Furthermore, the substrate may be one that exhibits low scattering at the aforementioned excitation light and fluorescence wavelengths. Examples of substrate materials include glass, synthetic quartz, quartz, borosilicate glass, etc. Alternatively, examples of materials include resins such as polystyrene, polypropylene, (meth)acrylic resin, polyamide, polyimide, melamine, ABS, polyphenylene oxide urethane, silicone, epoxy, and polydimethylsiloxane. Alternatively, films such as nitrocellulose and polyvinylidene fluoride, and gels such as agarose and acrylamide can be used as materials. These materials may be fixed or supported by a frame-like structure or another substrate as needed and used as an array plate.

[0019] Immobilization of biomaterials can be performed by known methods, such as using arrayers, stamps, semiconductor technology, or pipetting. Furthermore, to prevent substances contained in the sample from directly binding to the substrate, the array plate may be treated with a non-specific adsorption inhibitor containing a blocking agent.

[0020] <Biomote> In this specification, "biomolecule" refers to a substance immobilized on an array plate and contained in a spot. The biomolecule can be appropriately defined by the person carrying out the present invention according to the purpose, and anything can be a biomolecule. Biomolecule includes proteins, peptides, nucleic acids, small molecule compounds, viruses, cells, etc., and all of these may be of natural origin, synthesized, or genetically modified. More specifically, biomolecule includes antibodies, antigens, phosphorylated proteins, dephosphorylated proteins, small molecule compounds, receptors, enzymes, substrates, phosphorylation enzyme substrates, allergens, cytokines, hormones, bacteria, viruses, microorganisms, DNA, RNA, cDNA, cells, cell membrane components, cancer markers, disease markers, biologically derived substances, extracts from living organisms, blood, blood-derived substances, food, food-derived substances, natural products, natural product-derived substances, culture medium-derived substances, and any other substances.

[0021] <Specimen> The specimen can be appropriately determined by the person carrying out the present invention according to the purpose, and anything can be a specimen. Specimens include biological substances, extracts from living organisms, blood, blood-derived substances, food, food-derived substances, natural products, natural product-derived substances, culture medium-derived substances, etc. Substances contained in the specimen that are expected to react with biological substances are called targets. Targets include antibodies, antigens, phosphorylated proteins, dephosphorylated proteins, nucleic acids, small molecule compounds, receptors, enzymes, substrates, allergens, cytokines, hormones, bacteria, viruses, microorganisms, DNA, RNA, cDNA, cells, cell membrane components, cancer markers, disease markers, etc. By the method of this embodiment, interaction information between targets in the specimen and biological substances can be obtained, and information about the specimen can be obtained.

[0022] The sample may be pre-treated as appropriate, depending on the purpose and procedure. Treatments such as reduction and heat treatment may be performed. Reagents may also be added to the sample beforehand. A labeling substance may be bound to the target in the sample beforehand.

[0023] The sample may be in solid, liquid, or gaseous form, but it is used in liquid form after being diluted, suspended, or extracted in water, physiological saline, buffer solution, or other solutions as appropriate. The sample may contain surfactants, preservatives, non-specific adsorption inhibitors, and other additives. Reagents may also be added to the sample as needed.

[0024] <Labeling substance> To visualize the spots that have reacted with the target, the target or the spots that have reacted with the target are labeled; that is, a labeling substance is bound to the target, or a labeling substance is introduced to the spots that have reacted with the target. The labeling substance used is one that can be detected optically. Labeling substances include fluorescent substances, chemiluminescent substances, phosphorescent substances, dyes, gold nanoparticles, fluorescent particles, enzymes for enzymatic chemiluminescence or color reactions, and microparticles that absorb at specific wavelengths. The labeling substance may also include antibodies, ligands, or other binding sites. Binding or introduction of the labeling substance includes binding by hydrophobic interactions, electrostatic interactions, van der Waals interactions, hydrogen bonding, covalent bonding, PCR, introduction during synthesis, and other known labeling methods.

[0025] Typically, the labeling substance is a fluorescent substance. The first or second labeling step, described later, typically involves labeling with a fluorescent substance. Fluorescent substances are advantageous because they provide stable luminescence, do not require a substrate, can label regions specifically, are suitable for two-dimensional analysis, and allow for multiple labeling using fluorescent substances with different wavelengths. The type of fluorescent substance is appropriately selected according to the reaction system and optical system. Fluorescent substances include fluorescein, cyanine, rhodamine, Texas Red, coumarin dyes, fluorescent proteins, quantum dots, etc. Those skilled in the art can select a fluorescent substance with an appropriate excitation wavelength, fluorescence wavelength, and properties to suit the optical system and experimental setup.

[0026] Chemiluminescent materials are advantageous because they emit light upon the addition of a substrate, eliminating the need for an excitation light source, and they offer high sensitivity. When using chemiluminescent materials, an enzyme that catalyzes chemiluminescence or a substrate is used as a labeling substance. Substrates include enzymes such as alkaline phosphatase and horseradish peroxidase, as well as substrates containing acridinium. Chemiluminescence can generally be observed using a high-sensitivity CCD camera. On the other hand, a substrate or catalyst for luminescence needs to be supplied during detection. These are preferably added to the observation solution.

[0027] Gold nanoparticles have the property of absorbing light near specific wavelengths, depending on their particle size. When gold nanoparticles are used as a labeling material, the labeled spot can be detected by measuring the amount of light absorbed or reflected at a selective wavelength. Gold has a high affinity for sulfur, nitrogen, and oxygen atoms, and the object to be labeled is bonded to the surface of the gold nanoparticles through thiolation (addition of an -SH group) and sulfur-gold atom bonding.

[0028] <Observation solution> The observation solution is also referred to as a purging solution, in the sense that it replaces, or purges, optical background noise components. The observation solution is positioned to contact at least the spots on the array plate when acquiring optical information. The observation solution may also be positioned to contact part or all of the first surface of the array plate. The observation solution is preferably one that has good affinity with the solution used on the array plate in the preceding process. It is desirable that the observation solution has a refractive index close to that of the substrate of the array plate and can prevent oxidation of the material on the array plate, especially the labeling material. Furthermore, it is desirable that the observation solution does not fluoresce when irradiated with excitation light to acquire optical information. A glycerol solution is preferably used to satisfy these conditions, but it can be appropriately selected based on the properties of the labeling material, biological material, and sample.

[0029] The observation solution may also be used as a washing solution. Alternatively, the washing solution present on the array plate, the reagent used in the second labeling step, or any other solution used in the process may be used as the observation solution.

[0030] The observation solution has a refractive index that is at least closer to that of the array plate substrate than to that of air. Preferably, the observation solution is a liquid with a refractive index of 1.3 or higher, more preferably 1.33 to 1.60, and even more preferably 1.40 to 1.46, from the viewpoint of being closer to the refractive index of the array plate. Preferably, water, physiological saline, phosphate buffer, Tris-HCl buffer, other buffers, and other solutions containing 40 vol% to 90 vol% glycerol are examples. Glycerol is hygroscopic and has high viscosity, and if it exceeds 90 vol%, it becomes difficult to use. If it is less than 40 vol%, it deviates from the refractive index of the array plate. The observation solution may also optionally contain surfactants, preservatives, and non-specific adsorption inhibitors. Typically, the observation solution is a buffer containing 80 vol% glycerol. The observation solution may also contain a commercially available colorfastness inhibitor to prevent the fading of the dye. The colorfastness inhibitor may include Prolong, manufactured by ThermoFisher Scientific.

[0031] <Analysis method> In this embodiment, the analysis method may be partially or entirely performed manually by a person, but preferably, all processes are performed automatically by the apparatus. The method of this embodiment does not include a drying process. Therefore, it is advantageous that all processes are performed automatically by the apparatus, as there is no need to transport the array plate to a dryer. Conceptual diagrams of the processes in this embodiment are shown in Figures 1(a) and 1(b). Figure 1(a) shows the analysis method S1100 according to the first embodiment using a first labeling step, and Figure 1(b) shows the analysis method S2200 according to the second embodiment using a second labeling step. More specifically, it is described as follows. However, the present invention is not limited to the following description.

[0032] <<First Embodiment>> Figure 1(a) shows a process diagram of the analysis method S1100 according to the first embodiment. The analysis method S1100 includes a first labeling step S1100 which is performed before the sample introduction step S100 (described later), in which the sample is brought into contact with a plurality of spots and a portion of the plurality of spots is reacted with the target, and labels the target.

[0033] Sample introduction process (S100) This process involves bringing the sample and the spot into contact, causing some or all of the target to react with the biomolecules contained in the spot. These reactions include binding, chemical reactions, and enzymatic reactions between the target and the biomolecules. Reagents may be added to the sample as needed. At least enough sample is added to cover the spot. Part or all of the first surface of the array plate may be covered with the sample. The reaction between the substance in the sample and the biological material takes place at a temperature suitable for the reaction and for a desired time.

[0034] Furthermore, as a retention mechanism, the array plate can be provided with a frame, or the array plate can be placed in a suitable container. These frames or containers allow the sample, observation solution, replacement solution, reagents, and other liquids used in each of the following steps to be held on the array plate.

[0035] Sample volume reduction process (S200) This process reduces the amount of sample introduced in the sample introduction step S100. After this process, the targets that bound to the biomolecule in the sample introduction step S100 remain on the spot side, while targets that did not react, or targets that have undergone a chemical or enzymatic reaction with the biomolecule, are reduced. The sample on the spot is reduced by aspirating with a nozzle. The nozzle includes pipette tips, needles, pipettes, syringes, pumps, and combinations thereof. The sample may be reduced by tilting the array plate, or the container containing the array plate, relative to the horizontal. Alternatively, the sample may be reduced by decantation by simply tilting the array plate without using a nozzle, and then the liquid may be further reduced by aspirating with a nozzle. Reduction means reducing the volume to less than 10% of the original liquid volume. The same procedure applies not only to sample reduction but also to reduction of other liquids.

[0036] Observation solution introduction process (S300) This step brings the observation solution into contact with the spot. Performing this step eliminates the need for a drying step, thus leaving no drying marks and allowing the entire process from the sample introduction step S100 to the acquisition step S400 to proceed without interruption. The observation solution is added so as to at least cover the spot. It is sufficient for the spot to be in contact with the observation solution, but part or all of the first surface of the array plate may be covered with the observation solution. Preferably, the observation solution is in contact only with the first surface of the array plate, and the second surface is not in contact with the observation solution. Alternatively, the washing solution or other reagent solutions may be used directly as observation solutions, in which case the process of bringing them into contact corresponds to this process. Furthermore, the observation solution introduction step S300 can also serve as part of the sample volume reduction step S200. That is, by repeatedly reducing a portion of the sample on the spot, introducing the observation solution, and then reducing a portion of the mixture again, the sample volume can be reduced to less than 10% of the introduced volume, or to a desired level. With this method, it is possible to perform all steps while maintaining some kind of liquid on the spot at all times. Furthermore, the sample in contact with the spot can be replaced with the observation solution; that is, the introduced sample can be replaced with the observation solution without ever being replaced with any other solution.

[0037] Acquisition process (S400) This process acquires optical information about the spot. The optical information provides information about the presence or absence of the labeled substance, making it possible to determine whether or not the spot reacted with the target. Optical information of multiple spots is obtained when the spots are in contact with the observation solution. The information is acquired by an optical system, and the optical information includes at least one of the light intensity information and spectral information from the spots. The optical system is located on the side of the second surface of the array plate. If the labeling material is a fluorescent material, the optical system irradiates the spots with excitation light from the side of the second surface and detects the fluorescence emitted by the spots from the side of the second surface. The light intensity information and spectral information can be acquired in association with the addresses of the spots in the array plate. Based on the acquired optical information, further information about the sample can be acquired. This can be called the sample information acquisition process.

[0038] Because the refractive index of the observation solution is closer to that of the array plate compared to air, the light emitted from the optical system is less likely to be reflected at the interface between the array plate and the observation solution, resulting in optical information with a higher signal-to-noise ratio.

[0039] The optical system can be used without limitation as long as it can excite the fluorescent material and detect fluorescence from the spot, provided that the labeling material is fluorescent. An excitation light source is used to excite the fluorescent material, and examples of excitation light sources include laser light sources, light-emitting diodes, LEDs, mercury arcs, tungsten halogen lamps, etc. For detection, a CCD camera, photodiode, etc., may be used. The optical system may include filters as appropriate to irradiate or detect light of a limited wavelength. The optical system may also include lenses. The optical system may be scanning or non-scanning. A typical optical system is a confocal optical unit.

[0040] When chemiluminescent materials, gold colloids, dyes, etc., are used as labeling materials, an excitation light source is not required.

[0041] First marking process (S1000) In the analytical method S1100 according to the first embodiment and the analytical method S2200 according to the second embodiment described later, at least one of the first labeling step S1000 and the second labeling step S2000 is performed to label the spots. The first labeling step S1000 is performed when a labeling substance is bound to a target in the sample in advance before analysis. When the target to which the labeling substance is bound in the sample introduction step S100 binds to a biological substance, in the acquisition step S400, the spot to which the target is bound is labeled and observed, and it can be determined that a reaction has occurred. Methods for attaching the labeling substance to the target include chemical reactions via functional groups, PCR-based methods, kit-based methods, and all other known methods. This step is performed before the sample introduction step S100. A schematic of the process when the first labeling step S1000 is performed is shown in Figure 2(a). In the first labeling step S1000, the target 2000 is labeled with the labeling substance 2001 (Figure 2(a), top and second). In the sample introduction step S100 and the sample volume reduction step S200, the target reacts with the biomolecule 2002 in the spot 103, and the labeling substance 2001 is introduced into the spot 103 (Figure 2(a), third top and bottom). In the observation solution introduction step S300, the spot is brought into contact with the observation solution.

[0042] <<Second Embodiment>> Figure 1(b) shows a process diagram of the analysis method S2200 according to the second embodiment. The analysis method S2200 differs from the first embodiment S1100 in that it includes a second labeling step S2000 which is performed before the sample introduction step S200, which reduces the amount of sample in contact with multiple spots, and labels the target.

[0043] Second marking process (S2000) If the first labeling step S1000 is not performed, the second labeling step S2000 is performed. Both the first labeling step S2000 and the second labeling step S2000 may be performed. The second labeling step S2000 is performed after the sample volume reduction step S200 and labels the spots that have reacted with the target. The labeling substance is introduced specifically to the target bound to the spot, or to the change in biomolecules caused by the target. Through this step, the spots that have reacted with the target are labeled and observed, and it can be determined that a reaction has occurred.

[0044] Figure 2(b) shows a schematic of the process when the second labeling step S2000 is performed. Target 2000 reacts with spot 103 after the sample introduction step S100 (top of Figure 2(b)) and the sample volume reduction step S200 (second from the top of Figure 2(b)), causing a specific change 2003 in the biomolecule 2002 of spot 103. In the second labeling step S2000, the labeling substance 2001 is specifically introduced to the biomolecule change 2003 (third from the top of Figure 2(b)). An example of the biomolecule change 2003 is phosphorylation. The labeling substance is introduced via an antibody or ligand that recognizes the biomolecule change, or its secondary antibody (bottom of Figure 2(b)). Other known methods can also be used in this process. In the second labeling step S2000, the labeling substance is not introduced (fixed) to unreacted spots that did not react with the target. However, if left as is, the labeling substance remains unfixed on the substrate between the unreacted spots and the spots, resulting in background noise in the acquisition process. The spots are labeled by either the first labeling step S1000 or the second labeling step S2000, or both.

[0045] To complete the second labeling step S2000 so that the labeled substance is introduced to a portion of multiple spots, a cleaning step is performed to reduce unreacted spots and unfixed (unintroduced) labeled substance present on the substrate between spots from the first surface. The cleaning step reduces unreacted spots and unfixed labeled substance present on the substrate between spots by reducing the reagent solution 125-2 containing the labeled substance 2001 from one surface of the substrate (the first surface 101-1). In other words, the cleaning step selectively leaves the labeled substance introduced to the spots that reacted with the target on those spots by reducing the unfixed labeled substance present on the substrate between spots. The cleaning step described later may be referred to as part of the second labeling step S2000.

[0046] The residue on the first surface 101-1 of the reagent solution 125-2 used in the second labeling step S2000 is replaced with the washing solution used for washing in each washing step. Therefore, the washing solution can be rephrased as the replacement solution for the reagent solution 125-2. By replacing the washing solution multiple times, the residue on the first surface 101-1 of the reagent solution 125-2 decreases asymptotically towards 0. As will be described later, in embodiments where the washing solution is used as the observation solution, the observation solution is essentially a replacement solution that replaces the reagent solution 125-2. Bringing a predetermined component closer to 0 by liquid replacement can be rephrased as purging or purging, so the observation solution and washing solution can be rephrased as purging solutions.

[0047] Washing process This embodiment may include a washing step. The washing step is performed by placing a washing solution on a spot and reducing the amount of solution after washing. The washing step may be repeated. As the washing solution, water, physiological saline, phosphate buffer, Tris-HCl buffer, other buffers, and other solutions may be used, to which surfactants, preservatives, non-specific adsorption inhibitors, glycerol, and other additives may be added as appropriate. Typically, TBST, a Tris buffer containing polysorbate, may be used as the washing solution. The washing solution may be used as is as the observation solution. That is, the washing step may be included as part of the observation solution introduction step S300 described later.

[0048] <Detection of protein-protein interactions> One example of this embodiment is the observation of protein-protein interactions. As an example, a method for detecting antibodies in a sample using an array plate on which an antigen has been immobilized will be described below.

[0049] When detecting antibodies in a sample, the biomaterial used in the array plate is an antigen. After the first labeling step S1000, a fluorescent substance is bound to the antibodies in the sample. After the sample introduction step S100 described above, antibodies that exhibit a specific interaction with a predetermined antigen (biomaterial) contained in the relevant spot, among the antibodies contained in the sample, bind to the antigen at the relevant spot and are immobilized at the predetermined spot. A washing step may be performed between the sample volume reduction step S200, the observation solution introduction step S300, and the acquisition step S400. Alternatively, the sample introduction step S100 and the sample volume reduction step S200 may be performed without going through the first labeling step S1000. In that case, a second labeling step S2000 is performed. In the second labeling step S2000, a labeling substance is introduced to the target bound to the spot via a more specific substance. Both the first labeling step S1000 and the second labeling step S2000 may be performed. After that, an observation solution introduction step S300 and an acquisition step S400 are performed. A washing step is performed between each step as appropriate. The washing solution used in the washing step may be the observation solution.

[0050] Next, a method for detecting antigens in a sample using an array plate on which antibodies are immobilized is described below. In this case, after the sample introduction step S100 and the sample volume reduction step S200, a labeling substance is introduced in the second labeling step S2000. In the sample introduction step S100, the antigen in the sample binds to the antibody immobilized at the relevant spot and is immobilized at that spot. Here, the relevant spot refers to a spot containing an antibody as a biomaterial that specifically interacts with the antigen in the sample. In the second labeling step S2000, the labeling substance may be introduced using an antibody that recognizes the antigen. The antibody used at this time may be the same antibody as the antibody used as a biomaterial, or an antibody with a different recognition site from the antibody used as a biomaterial may be used. In this specification, the elementary processes of each step may be explained by focusing on a spot that specifically interacts with the target in the sample, the antibody in the reagent, and the reaction promoting component, and such a spot of focus may be referred to as the relevant spot. Also, a spot containing a biomaterial that specifically interacts with the target in the sample may be referred to as the relevant spot.

[0051] <Nucleic acid detection> One embodiment of this method is the detection of nucleic acids in a sample. When detecting nucleic acids in a sample, the biomaterial in the array plate is synthetic oligoDNA. A labeling substance is bound to the nucleic acids in the sample via a first labeling step S1000. The synthetic oligoDNA has a sequence complementary to the target DNA sequence. In the sample introduction step S100, a hybridization reaction is performed. A sample volume reduction step S200, an observation solution introduction step S300, and an acquisition step S400 are performed. A washing step is performed as needed. The washing solution in the washing step may be an observation solution.

[0052] <Detection of phosphorylation enzymes> One embodiment of this method involves detection using phosphorylation enzymes in the sample. The method for detecting phosphorylation enzymes in the sample is described below. If a phosphorylation enzyme is detected, the biomaterial on the array plate is a phosphorylation enzyme substrate. A phosphorylation enzyme substrate refers to a substrate that is phosphorylated by a phosphorylation enzyme, and is a sub-concept of protein or peptide. In the sample introduction step S100, the substrate at the relevant spot is phosphorylated by the phosphorylation enzyme contained in the sample. Here, the relevant spot refers to a spot that contains a phosphorylation enzyme substrate as biomaterial that undergoes a specific phosphorylation reaction by the phosphorylation enzyme in the sample. After the sample volume reduction step S200, the second labeling step S2000 is performed. In the second labeling step S2000, the labeling substance is introduced to the relevant spot via a phosphorylation site recognition substance. A phosphorylation site recognition substance is a substance that specifically recognizes the phosphorylated site on a substrate that has been phosphorylated by a phosphorylation enzyme. Antiphosphorylated amino acid antibodies are given as examples of phosphorylation site recognition substances. If the labeling substance is not bound to the phosphorylation site recognition substance, the labeling substance is further introduced via a specific reaction, including a secondary antibody or a biotin avidin reaction. Subsequently, the observation solution introduction step S300 and the acquisition step S400 are performed. A washing step is performed as necessary. The washing solution used in the washing step may also be the observation solution. The reagent solution used in the second labeling step S2000 contains at least a primary antibody containing a phosphorylation site recognition substance that recognizes phosphorylation sites, and optionally a secondary antibody containing a labeling substance.

[0053] <Device> Furthermore, as one embodiment of the present invention, an analytical apparatus for analyzing a sample containing a target using an array plate having a plurality of spots containing biological material on one surface, comprising: a sample introduction mechanism for bringing the sample into contact with the plurality of spots; a sample volume reduction mechanism for reducing the amount of the sample in contact with the plurality of spots; an observation solution introduction mechanism for bringing the surface into contact with the observation solution so that the plurality of spots are in contact with the observation solution; and an acquisition mechanism for acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, wherein the analytical apparatus comprises at least one of: a first labeling mechanism that operates before the sample introduction mechanism operates and labels the target; and a second labeling mechanism that operates after the sample volume reduction mechanism operates and labels the spots that have reacted with the target.

[0054] The apparatus according to this embodiment is described in Figure 3(a). The apparatus has a sample introduction mechanism 3001, a sample volume reduction mechanism 3002, an observation solution introduction mechanism 3003, and an acquisition mechanism 3004, and further includes at least one of a first labeling mechanism 3005 and a second labeling mechanism 3006. The sample introduction mechanism 3001, the sample volume reduction mechanism 3002, and the observation solution introduction mechanism 3003 are mechanisms for performing the steps described above in the sample introduction step S100, the sample volume reduction step S200, and the observation solution introduction step S300, respectively. The first labeling mechanism 3005 and the second labeling mechanism 3006 are mechanisms for performing the steps described above in the first labeling step and the second labeling step. The apparatus may further have a holding mechanism for holding the liquid containing the observation solution on the array plate. The holding mechanism may be located on the array plate or provided separately from the array plate. Examples of the holding mechanism include a frame provided on the array plate or a container in which the array plate can be housed.

[0055] Furthermore, the device may have a mechanism to tilt the array plate or the holding mechanism relative to the horizontal plane in order to facilitate the suction of liquid from the holding mechanism. The tilting mechanism may be configured to push the array plate or the holding mechanism vertically downward. The array plate can be tilted relative to the horizontal plane by being pushed by the tip of a nozzle. Specifically, the array plate or the holding mechanism can be pushed with either a pipette tip or a needle. As part of the tilting mechanism, an elastic body such as a leaf spring or a torsion spring may be included in the base that supports the array plate or the holding mechanism.

[0056] To facilitate liquid aspiration, the holding mechanism may be designed to have a lowest point. Having a lowest point means that when the holding mechanism is tilted by the mechanism described above, one of its vertices or a part of it is positioned lower than the other points. With this design, when tilted, the liquid gathers in one place and is easily aspirationed. In order to have a lowest point, the shape of the bottom surface of the mechanism that holds the observation liquid can be set to any shape, taking into consideration the ease of liquid aspiration. For example, the shape can be not only a common rectangle, but also a triangle, pentagon, other polygons, a sector, a circle, an ellipse, or any other shape.

[0057] Furthermore, the present invention also includes, as one of its further embodiments, A mounting section on which an array plate having multiple spots containing biological material on one side is placed, A holding portion that holds the liquid so that it is in contact with one of the aforementioned surfaces, A liquid supply / drainage unit that enables the introduction of the liquid into the liquid-holding region of the holding unit, or the discharge of the liquid held by the holding unit, A moving part that moves the liquid supply and drainage part relative to the previously described mounting part, An optical system that irradiates light onto the plurality of spots and detects the light from the plurality of spots, A scanning unit that moves the optical system relative to the previously described mounting unit, The present invention provides an analytical apparatus comprising: a sample introduction step of bringing a sample into contact with the plurality of spots and reacting a portion of the plurality of spots with a target contained in the sample; a sample volume reduction step of reducing the amount of the sample in contact with the plurality of spots; an observation solution introduction step of bringing the surface into contact with the observation solution so that the plurality of spots are in contact with the observation solution; and an acquisition step of acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution; and a control unit that controls the time at which each of these steps is performed, wherein the control unit controls the time at which at least one of the following steps is performed: a first labeling step performed before the sample introduction step to label the target, and a second labeling step performed after the sample volume reduction step to label the spots that have reacted with the target.

[0058] In this embodiment, the control unit can control the mounting unit, the liquid supply and drainage unit, the moving unit, the optical system, and the scanning unit in order to perform the sample introduction step S100, the sample volume reduction step S200, the observation solution introduction step S300, and the first labeling step S1000. In this case, the control unit may, in parallel with the execution of the acquisition process for the first array plate, perform at least one of the following steps for a second array plate different from the first array plate: the sample introduction step S100, the sample volume reduction step S200, the observation solution introduction step S300, and the first labeling step S1000.

[0059] Furthermore, in this embodiment, the control unit can control the mounting unit, the liquid supply and drainage unit, the moving unit, the optical system, and the scanning unit in order to perform the sample introduction step S100, the sample volume reduction step S200, the observation solution introduction step S300, and the second labeling step S2000. In this case, the control unit may, in parallel with the execution of the acquisition process for the first array plate, perform at least one of the following steps for a second array plate different from the first array plate: the sample introduction step S100, the sample volume reduction step S200, the observation solution introduction step S300, and the second labeling step S2000.

[0060] The analytical apparatus according to this embodiment is described in Figure 3(b). The mounting section 1001 is for mounting an array plate having a plurality of spots containing biological material on its first surface. The holding section 1015 holds the liquid so as to be in contact with at least the first surface of the array plate. The holding section 1015 may be attached to the array plate, or it may be provided separately from the array plate, or it may be provided on the mounting section 1001.

[0061] The liquid supply and discharge section 1002 introduces a predetermined liquid into the holding section 1015 or discharges the liquid held by the holding section 1015. The liquid supply and discharge section 1002 may include a nozzle capable of aspirating and discharging liquids, and may further include a sample bottle for storing a sample, a reagent bottle for storing a reagent solution, and an observation solution bottle for storing an observation solution. A nozzle is a general term for an instrument used to aspirate and discharge liquids. Nozzles include pipette tips, needles, pipettes, syringes, pumps, and combinations thereof. However, when discharging liquid as waste, it is preferable to use a vacuum pump in combination to maintain negative pressure in the waste bottle to prevent backflow of the waste liquid. When a vacuum pump is used, the waste liquid is discharged using a solenoid valve pipeline as needed.

[0062] The movable part 1013 moves the liquid supply / drainage part 1002 relative to the mounting part 1001. An example of a movable part is a stage for moving a nozzle.

[0063] The optical system 1003 irradiates a spot on the array plate 100 of the mounting unit 1001 with excitation light and detects the resulting fluorescence. The scanning unit 1014 scans the optical system 1003, the mounting unit 1001, or a part thereof, to observe the optical information of the entire required area of ​​the array plate when the observable area of ​​the optical system 1003 is limited. The scanning unit 1014 may scan the mounting unit 1001, the optical system 1003, only a part of the optical system 1003, or only the excitation light source. It is preferable that the optical system 1003 is located on the side of the array plate 100 that is different from the first surface 101-1, so that interference between the optical system 1003 and the nozzles included in the liquid supply / drainage unit 1002 can be avoided, space can be used effectively, and the entire device can be miniaturized. Furthermore, moving relative to the mounting section 1001 means moving in order to introduce or discharge liquid into or out of the array plate mounted on the mounting section 1001, or to acquire optical information of the spots on the array plate mounted on the mounting section 1001, so as to bring the liquid supply / drainage section 1002 or optical system 1003 closer to the mounting section, or to bring the liquid supply / drainage section 1002 or optical system 1003 further away from the mounting section 1001.

[0064] The control system 1004 controls at least the order in which the processes related to the components constituting the analytical apparatus 4001 are executed. The components constituting the analytical apparatus 4001 include a mounting unit 1001, a liquid supply and drainage unit 1002, a moving unit 1013, an optical system 1003, and a scanning unit 1014.

[0065] Furthermore, the control system 1004 controls at least the order in which the steps related to the components constituting the analytical method are executed. The analytical method is an analytical method including a first labeling step, as shown in Figure 1(a), which includes a first labeling step S1000, a sample introduction step S100, a sample volume reduction step S200, an observation solution introduction step S300, and an acquisition step S400, and an analytical method including a second labeling step S2000, as shown in Figure 1(b), which includes a sample introduction step S100, a sample volume reduction step S200, a second labeling step S2000, an observation solution introduction step S300, and an acquisition step S400. The analytical method may include at least one of the first labeling step S1000 and the second labeling step S2000, or it may include both. Therefore, the control system 1004 may be referred to as a sequencer or a control unit. The control system 1004 controls at least the time at which the processes related to the components constituting the analysis method are executed.

[0066] The control system 1004 can control the aforementioned placement unit, the supply and drainage unit, the moving unit, the optical system, and the scanning unit in order to perform the sample introduction step S100, the sample volume reduction step S200, the observation solution introduction step S300, and the first labeling step S1000. In this case, the control unit may, in parallel with the execution of the acquisition step S400 for the first array plate, perform at least one of the following steps with respect to a second array plate different from the first array plate: the sample introduction step S100, the sample volume reduction step S200, the observation solution introduction step S300, and the first labeling step S1000.

[0067] Furthermore, in this embodiment, the control system 1004 can control the mounting unit 1001, the supply and drainage unit 1002, the moving unit 1013, the optical system 1003, and the scanning unit 1014 in order to perform the sample introduction step S100, the sample volume reduction step S200, the observation solution introduction step S300, and the second labeling step S2000. In this case, the control unit may, in parallel with the execution of the acquisition step S400 for the first array plate, perform at least one of the following steps with respect to a second array plate different from the first array plate: the sample introduction step S100, the sample volume reduction step S200, the observation solution introduction step S300, and the first labeling step S1000.

[0068] The control system 1004 has computer functionality. The control system 1004 may be integrated with a desktop PC (Personal Computer), laptop PC, tablet PC, smartphone, etc. The control system 1004 controls the mounting unit 1001, the liquid supply and drainage unit 1002, the moving unit 1013, the optical system 1003, and the scanning unit 1014 so that each step of an analytical method can be performed, which includes a sample introduction step of bringing a sample into contact with multiple spots and reacting a portion of the multiple spots with a target, a sample volume reduction step S200 of reducing the amount of sample in contact with the multiple spots, an observation solution introduction step S300 of bringing the first surface of the array plate into contact with the observation solution so that the multiple spots are in contact with the observation solution, and an acquisition step of acquiring optical information of the multiple spots while the multiple spots are in contact with the observation solution, and further includes at least one of a first labeling step S1000 of labeling the target before the sample introduction step, and a second labeling step S2000 performed after the sample volume reduction step S2000 of labeling the spots that have reacted with the target.

[0069] The control system 1004 includes a CPU 1005, RAM 1006, ROM 1007, and HDD 1008 to realize the functions of a computer that performs calculations and storage. CPU stands for Central Processing Unit, RAM for Random Access Memory, ROM for Read Only Memory, and HDD for Hard Disk Drive. The control system 1004 also includes a communication interface 1009, a display device 1010, and an input device 1011. The CPU 1005, RAM 1006, ROM 1007, HDD 1008, communication interface 1009, display device 1010, and input device 1011 are interconnected via a bus 1012. The display device 1010 and input device 1011 may also be connected to the bus 1012 via a drive device (not shown) for driving these devices.

[0070] In Figure 3(b), the components of the control system 1004 are shown as a single integrated device; however, some of these functions may be provided by external devices. The display device 1010 and the input device 1011 may be external devices separate from the components that constitute the functions of the computer, including the CPU 1005.

[0071] The CPU 1005 performs predetermined operations according to programs stored in the RAM 1006, HDD 1008, etc., and also has the function of controlling each part of the control system 1004.

[0072] RAM 1006 is composed of a volatile storage medium and provides a temporary memory area necessary for the operation of CPU 1005. ROM 1007 is composed of a non-volatile storage medium and stores the necessary information for the program used in the operation of the control system 1004. CPU 1005 loads the program stored in ROM 1007 into RAM 1006 and executes it to realize the functions of the mounting unit 1001, the fluid supply and drainage unit 1002, and the optical system 1003. HDD 1008 is composed of a non-volatile storage medium and is a storage device that stores information regarding the number and position of spots on the array plate, light intensity information, or spectral information.

[0073] Communication I / F 1009 is a communication interface based on standards including Wi-Fi® and 4G, and is a module for communicating with other devices. Display device 1010 is a liquid crystal display, OLED (Organic Light Emitting Diode) display, etc., and is used for displaying videos, still images, text, etc. Input device 1011 is a button, touch panel, keyboard, pointing device, etc., and is used by the user to operate the control system 1004. Display device 1010 and input device 1011 may be integrally formed as a touch panel.

[0074] In addition to the configuration shown in Figure 3(b), other devices may be added, or some devices may be omitted. Furthermore, some devices may be replaced with other devices having similar functions. Moreover, some functions may be provided by other devices via a network, and the functions constituting this embodiment may be distributed and implemented across multiple devices. The HDD 1008 may be replaced with an SSD (Solid State Drive) using semiconductor elements including flash memory, or it may be replaced with cloud storage.

[0075] Furthermore, the control system 1004 can control operations from the optical system 1003 and the fluid supply / drainage unit 1002 in parallel. That is, in parallel with the execution of the acquisition process for the first array plate, the control system can execute and control processes for the second array plate that include at least one of the following: the sample introduction process, the sample volume reduction process S200, the observation solution introduction process S300, the first labeling process S1000, and the second labeling process S2000. This allows for simultaneous analysis of multiple array plates, saving analysis time.

[0076] <Program> As one embodiment, the present invention provides a program for a control system in an analytical apparatus to perform the series of controls described above. It also provides a program for causing a computer to execute the above-described method.

[0077] Embodiments of the present invention will be described below with reference to the figures. However, the present invention is not limited to the following description. The analytical apparatus 4001 and its desktop arrangement according to this embodiment will be explained using Figures 4(a) and 4(b).

[0078] In Figure 4(a), 101 is a substrate, specifically a glass slide, and 103 is a spot. One type of biological material is fixed to each spot. The array plate 100 comprises the substrate 101 and the spots 103. 105 is a frame that serves as a holding mechanism provided on the array plate 100, and is an example of a holding part 1015. The observation solution 107 is held inside the frame 105.

[0079] The pipette operating section 117 is attached to the vertical stage 113 via the pipette support section 115. The vertical stage 113 is attached to the horizontal stage 111. The vertical stage 113 is movable in the vertical direction, and the horizontal stage 111 is movable in the horizontal direction. Therefore, the pipette support section 115, the pipette operating section 117, the pipette tip attachment / detachment section 119, and the pipette tip 151-1 are movable in the vertical and horizontal directions. Note that the pipette operating section 117 and pipette tip attachment / detachment section 119 are examples of the liquid supply / drainage section 1002, and the vertical stage 113 and horizontal stage 111 are examples of the moving section 1013. 121 is a pipette tip rack, showing the state in which unused pipette tips (151-2, 151-3, ...) are placed.

[0080] 123 (123-1, 123-2) is a reagent solution bottle for holding the reagent solution, and 124 is an observation solution bottle for holding the observation solution. 125 (125-1, 125-2) is the reagent solution, and 107 is the observation solution. The reagent solution refers to the liquid added to the sample, and in this embodiment, it includes a reaction solution, a reaction stop solution, a washing solution, and a labeling substance. 141 is a sample bottle, and 143 is a sample. 127 is a waste liquid bottle, and 153 is waste liquid. 129 is a waste chip box, and 155 is waste chips.

[0081] In Figure 4(a), two reagent solution bottles are shown for convenience, but in reality, you should prepare an appropriate number as needed. Similarly, three pipette tips are shown for convenience, but in reality, you should prepare the number you need as appropriate.

[0082] 221 is a confocal optical unit and is an example of the optical system 1003. The optical unit 221 is mounted on the horizontal stage 223 and is movable in the horizontal direction. The horizontal stage 223 is an example of the scanning unit 1014.

[0083] In this embodiment, an example in which a fluorescent substance is used as the labeling substance will be described. The optical unit 221 is located on the side of the second surface of the array plate 100. The components constituting the optical unit 221 are described below. 201 is a semiconductor laser that emits light with a wavelength of 670 nm, 203 is a collimating lens, 205 is a bandpass filter that transmits light near the wavelength of 670 nm, 207 is a longpass filter with a cut-on wavelength of 685 nm, and 209 is an objective lens. Light emitted from the semiconductor laser passes through the collimating lens 203 and the bandpass filter 205, is reflected by the longpass filter 207, and is focused by the objective lens 209 onto the spot 103 on the first surface 101-1 of the array plate 100.

[0084] Furthermore, 211 is a bandpass filter that transmits light near a wavelength of 716 nm, 213 is an imaging lens, 215 is a pinhole, and 217 is a photomultiplier tube. Light emitted from the spot passes through the objective lens 209 and the long-pass filter 207, and is focused by the imaging lens 213. Light that has passed through the pinhole 215 is detected by the photomultiplier tube 217.

[0085] The horizontal stage 223 scans the optical unit 221 along the array plate 100, thereby acquiring two-dimensional images of multiple spots on the array plate 100.

[0086] According to this embodiment, the observation liquid 107 is held in a holding mechanism provided on the array plate 100, and optical information of the spot 103 is obtained while the spot 103 is in contact with the observation liquid.

[0087] Figure 4(b) shows the analytical apparatus according to this embodiment arranged on a desk. In Figure 4(b), 4001 is a sample analyzer. The analyzer 4001 is placed on a workbench 4101. The analyzer 4001 according to this embodiment is equipped with a door 4003 that opens and closes a port for inserting and removing reagents into and out of the device. An interlock mechanism may be provided that stops the dispensing stage or stops the emission of the laser light source if the door 4003 is opened while the analyzer 4001 is in operation. The analyzer 4001 is equipped with an emergency stop switch 4005 that is activated when the operator recognizes any other abnormality. 4051 is a liquid crystal display, 4053 is a keyboard, and 4055 is a mouse. These are input units that send measurement conditions and higher-level commands from the operator to a control unit (not shown). The control unit may be built into the analyzer 4001, or it may be located on a local PC or a cloud-based computer. The local PC may be placed on or below the workbench 4101. The control unit can control the timing of each step in the sample introduction process S100, the sample volume reduction process S200, the observation solution introduction process S300, and the acquisition process, and furthermore, According to this embodiment, it is possible to perform sample analysis in an environment with good workability. [Examples]

[0088] The analytical method according to the present invention is described. Here, an example is given in which the purpose is to analyze a phosphorylation pathway, using a cell extract as the sample and targeting a phosphorylation enzyme. The array plate has spots containing biomolecules, which are proteins that are substrates whose phosphorylation reaction is specifically catalyzed by phosphorylation enzyme substrates. A fluorescent substance is used as the labeling substance. Figures 5 to 7 illustrate the process steps in Example 1.

[0089] (1) Preparation (Figure 5(a)) An array plate 100 is placed in a predetermined position on a substrate 101, in which spots 103, each immobilized with multiple types of proteins, are arranged in an array shape, and a frame 105, which serves as a holding mechanism, is provided around them. A pipette tip rack 121 equipped with unused pipette tips 151 (151-1, 151-2, 151-3, ...) is placed in its designated position. For convenience, three pipette tips are shown in the diagram. Reagent solution bottles 123 (123-1, 123-2...) that hold the reagent solutions 125 (125-1, 125-2...) necessary for the reaction, and observation solution bottles 124 that hold the observation solution 107 are placed in their designated positions. Although two reagent solution bottles are shown in the diagram for convenience, in reality, only the necessary number are used. Reagent solution 125-1 in this example contains phosphorylation-promoting component 125-10. Reagent solution 125-2 is a solution containing a fluorescent substance, a fluorescent dye (Alexa Flour 680)-conjugated secondary antibody. The fluorescent dye-conjugated secondary antibody corresponds to labeling substance 2001. Observation solution 107 is a glycerol solution. In addition, although not shown in the diagram, drug containers may be set up to supply other necessary liquids such as phosphorylation reaction stop solution, TBST, primary antibody solution, colorfastness inhibitor, and pure water. Furthermore, a sample bottle 141 containing the sample 143 to be analyzed is placed in a predetermined position. The sample 143 contains the target 2000. In this example, the sample is obtained by lysing human-derived model cells after culturing them. An empty waste liquid bottle 127 and a waste chip box 129 are installed.

[0090] (2) Attach the pipette tip (Figure 5(b)) The horizontal stage 111 and the vertical stage 113 are controlled to attach the pipette tip 151-1 to the pipette tip attachment / detachment section 119.

[0091] (3) Sample inhalation (Figure 5(c)) The horizontal stage 111 and vertical stage 113 are controlled to move the pipette tip 151-1 into the sample bottle 141, and the pipette operating unit 117 is controlled to aspirate the sample 143.

[0092] (4) Mixing of the sample and phosphorylation reaction solution (Figure 6(d)) Under the control of the pipette operating unit 117, the sample 143 is drawn in and discharged into the reagent solution bottle 123-1. Reagent solution 145 is a mixture of reagent solution 125-1 containing phosphorylation-promoting component 125-10 and the sample 143. If necessary, the mixture of the reagent solution and the sample is stirred using the pipette tip 151-1. Note that since the reaction between the sample and the phosphorylation-promoting component may progress over time after mixing, it is preferable to mix them immediately before contact with the spot.

[0093] (5) Reagent solution inhalation (Figure 6(e)) Under the control of the horizontal stage 111 and the vertical stage 113, the pipette tip 151-1 moves into the reagent solution bottle 123-1, and under the control of the pipette operating unit 117, the reagent solution 145 is drawn in.

[0094] (6) Reagent solution supply (Figure 6(f)) Control of the horizontal stage 111 and the vertical stage 113 moves the pipette tip 151-1 onto the array plate 100, and control of the pipette operating unit 117 supplies the reagent solution 145 onto the spot 103.

[0095] (7) Phosphorylation reaction (not shown) Array plate 100 is left to stand for approximately 5 hours to promote the phosphorylation reaction.

[0096] (8) Reduction of reagent solution (Figure 7(g)) Controlled by the horizontal stage 111 and the vertical stage 113, the tip of the pipette tip 151-1 moves directly above the array plate 100, and controlled by the pipette operating unit 117, the reagent solution 145 is drawn in.

[0097] (9) Disposal of pipette tips (Figure 7(h)) Under the control of the horizontal stage 111 and the vertical stage 113, the pipette tip 151-1 moves onto the waste liquid bottle 127, and under the control of the pipette operating unit 117, the reagent solution 145 is discharged, and then the tip moves onto the waste tip box 129 for disposal.

[0098] (10) Termination of phosphorylation reaction (not shown) The phosphorylation reaction cessation solution is supplied and reduced using the same methods as in (2), (5), (6), (8), and (9).

[0099] (11) Cleaning (not shown in the illustration) For cleaning purposes, TBST is supplied and reduced using the same methods as in (2), (5), (6), (8), and (9).

[0100] (12) Primary antibody introduction (not shown in the illustration) Using the same method as in (2), (5), (6), (8), and (9), the primary antibody solution is supplied, left to stand for 1 hour, and the primary antibody solution is reduced, and the primary antibody is introduced into the phosphorylated protein.

[0101] (13) Cleaning (not shown in the illustration) For cleaning purposes, TBST is supplied and reduced using the same methods as in (2), (5), (6), (8), and (9).

[0102] (14) Secondary antibody introduction (Figure 7(i)) Using the same method as in (2), (5), (6), (8), and (9), reagent solution 125-2, which is a fluorescent dye-conjugated secondary antibody solution, is supplied using pipette tip 151-2, allowed to stand for 1 hour, and then the amount of reagent solution 125-2 is reduced. Through this process, the secondary antibody, to which the fluorescent dye is conjugated, is introduced to the phosphorylated protein, and the introduction of the labeling substance to the spots containing the protein that has reacted with the phosphorylation enzyme in the sample is completed.

[0103] (15) Cleaning (not shown in the illustration) For cleaning purposes, TBST is supplied and reduced using the same methods as in (2), (5), (6), (8), and (9).

[0104] (16) Supply of observation solution (Figure 8(j)) The observation solution 107 is supplied using the same method as in (2), (5), and (6).

[0105] Next, the fluorescence detection process is explained using Figure 9. Figure 9 is a diagram illustrating the optical detection process. The inside of the frame 105 of the array plate 100 is filled with observation solution 107. Light emitted from the semiconductor laser 201 passes through the collimating lens 203 and the bandpass filter 205, is reflected by the longpass filter 207, passes through the objective lens 209, and is focused onto the spot 103 on the first surface 101-1 of the array plate 100.

[0106] Spots containing proteins that have reacted with phosphorylation enzymes in the sample emit fluorescence because a fluorescent substance has been introduced into them. The fluorescence emitted from spot 103 passes through the objective lens 209 and the long-pass filter 207 and is focused by the imaging lens 213. The light that passes through the pinhole 215 is detected by the photomultiplier tube 217.

[0107] The optical unit 221 is scanned along the array plate 100 by the horizontal stage 223, thereby acquiring a two-dimensional fluorescence image of the array plate 100.

[0108] In this embodiment, a glycerol solution with a refractive index of 1.4 or higher, close to that of glass, is used as the observation solution. Therefore, compared to cases where no observation solution is used, stray light due to reflected light is reduced, and the signal-to-noise ratio can be improved. Furthermore, the process from bringing the sample into contact with multiple spots to acquiring optical information of the multiple spots while they are in contact with the observation solution can be carried out continuously without a drying process, which is expected to improve throughput. In addition, the problems caused by uneven drying are suppressed.

[0109] The structure of the frame, which is the holding mechanism, will be explained using Figure 10. In the figure, (a-1), (b-1), and (c-1) are top views of the array plate 100, and (a-2), (b-2), and (c-2) are cross-sectional views of the dotted circled areas in the corresponding top views.

[0110] As shown in Figures 10(a-1) and 10(a-2), a rectangular rubber 501 and a frame 503 with a concave portion that contacts the rubber 501 are attached to the array plate 100. Furthermore, as shown in Figures 10(b-1) and 10(b-2), a retaining member 505 is fitted to the side wall so as to compress the rubber between the frame 503 and the substrate 101, thereby forming a frame. The frame 503 may be provided with a recess to facilitate the fitting of the retaining member 505. The frame structure is not limited to this; any shape is acceptable as long as it can hold the liquid. [Examples]

[0111] This section describes the case where the target of the sample is an antigen, and the presence or absence of the antigen is to be determined and / or quantified. In this case, it is effective to use an array plate having spots containing antibodies that specifically identify the antigen as biological material, hereinafter also referred to as an antibody array plate. Prepare the sample extracted from the living organism, along with reagent solutions including a biotinylated antibody cocktail containing fluorescently labeled streptavidin, a washing solution, and an observation solution.

[0112] The reaction process is as follows: First, by placing the sample in contact with the first surface of the antibody array plate and leaving it for a predetermined time, the antibodies in the antibody array plate react with the specific antigen. Next, after washing, a cocktail of biotinylated detection antibodies, in which biotin is bound to antibodies specific to the antigen, is placed on the first face of the antibody array plate. Furthermore, after washing, fluorescently labeled streptavidin, a fluorescent substance conjugated with streptavidin which has the property of binding to biotin, is supplied to the array plate, and the fluorescent substance is introduced to the spots that react with the target. Finally, the liquid on the first surface of the array plate is replaced with the observation solution, and optical information is obtained, as in Example 1. This embodiment enables efficient detection and quantification of the presence or absence of a target in a sample.

[0113] Similar to Example 1, in this example, a glycerol solution with a refractive index of 1.4 or higher, close to that of glass, is used as the observation solution. Therefore, compared to cases where no observation solution is used, stray light due to reflected light is reduced, and the signal-to-noise ratio can be improved. In addition, the process from bringing the sample into contact with multiple spots to acquiring optical information of the multiple spots while they are in contact with the observation solution can be carried out continuously without a drying process, which is expected to improve throughput. Furthermore, the problems caused by uneven drying are minimized. [Examples]

[0114] To obtain information about the immune system of a specimen, an array plate having spots containing multiple antigen molecules as biomaterials, hereinafter also referred to as an antigen array plate, is used. If antibodies specific to the antigen at the spot are present in the sample, those antibodies will react with the antigen. In this example, the sample is human serum, and the target is IgG antibody. The sample, along with reagent solutions including a biotinylated anti-human IgG antibody cocktail, a reagent solution containing fluorescently labeled streptavidin, a washing solution, and an observation solution, are prepared.

[0115] The process is as follows: First, the sample is placed in contact with the first surface of the antigen array plate for a predetermined time, allowing the antigens on the antigen array plate to react with the antibodies in the sample. Next, after washing, the biotinylated anti-human IgG antibody cocktail is placed on the first surface of the antigen array plate, and secondary antibodies bind to the antibodies that have bound to the antigen. Furthermore, after washing, fluorescently labeled streptavidin, which has the property of strongly binding to biotin, is supplied, and the secondary antibody is labeled. Finally, the liquid on the antigen array plate is replaced with the observation solution, and fluorescence detection is performed in the same manner as in Example 1.

[0116] This embodiment makes it possible to efficiently assess the immune status of a sample. Similar to Example 1, in this example, a glycerol solution with a refractive index of 1.4 or higher, close to that of glass, is used as the observation solution. Therefore, compared to cases where no observation solution is used, stray light due to reflected light is reduced, and the signal-to-noise ratio can be improved. In addition, the process from bringing the sample into contact with multiple spots to acquiring optical information of the multiple spots while they are in contact with the observation solution can be carried out continuously without a drying process, which is expected to improve throughput. Furthermore, the problems caused by uneven drying are minimized. [Examples]

[0117] When analyzing the activity of protein kinases, protein array plates are used, which are array plates that have spots containing the protein that serves as the substrate for protein kinase as a biomaterial. Alternatively, peptide array plates are used, which are array plates that have spots containing peptides with the sequence of the substrate site immobilized as a biomaterial. The process and effects in this case are as described in Example 1. [Examples]

[0118] The optical system related to the analytical apparatus according to the present invention will be explained with reference to Figure 11. Figure 11(a) shows the same configuration as described in Example 1, where fluorescence is detected from the back side of the slide glass, which is the second surface side of the array plate. The optical system configuration is not limited to this, and various forms can be taken as shown below. Figure 11(b) shows that the objective lens 309 is positioned on the first surface 101-1 side of the array plate 100, i.e., on the surface side of the substrate 101, and fluorescence is detected from the same side. In this arrangement as well, stray light due to reflected light can be reduced compared to when no observation solution is used, thus improving the signal-to-noise ratio. Furthermore, Figure 11(c) shows an array plate 100 placed inside a transparent container 401, which is filled with observation solution 107. 409 is an objective lens that detects fluorescence transmitted through the substrate 101 and the container 401. In this configuration as well, stray light due to reflected light can be reduced compared to the case without observation solution, thus improving the signal-to-noise ratio. In addition, since there is no need to provide a frame on the array plate 100, there is an advantage that spots can be placed and acquired on the slide glass without geographical limitations, compared to Figures 11(a) and (b). Furthermore, as shown in Figure 11(d), fluorescence may also be detected from the side of the first surface 101-1 of the array plate. [Examples]

[0119] Figures 12 and 13 illustrate a method for reducing reagent solution volume using a substrate tilting mechanism related to a sample analyzer according to the present invention.

[0120] Figure 12 illustrates the support base for supporting the array plate. Figure 12(a) is a top view, where 601 is the support part, 603 is the array plate support part, 605 is the leaf spring, and 607 is the retaining part. Note that the support base or array plate support part 603 in this figure or subsequent figures is an example of the mounting part 1001. Figure 12(b) is a cross-sectional view of part AA, and Figure 12(c) is a cross-sectional view of part BB.

[0121] Figure 13 illustrates the method of discharging the liquid. Figure 13(a) shows the array plate support 603 supporting the array plate 100 containing the reagent solution 126. The vertical stage (not shown) is controlled to move the pipette tip 151 downwards, and the tip of the pipette tip 151 pushes against the inside of the frame 105. As a result, the array plate 100 tilts, as shown in Figure 13(b). Next, as shown in Figure 13(c), the pipette control unit (not shown) is controlled so that the pipette tip 151 draws in the reagent solution 126. Subsequently, the pipette tip 151 moves upward by controlling the vertical stage (not shown). At that time, the array plate 100 returns to its original position due to the spring force of the leaf spring 605. According to this embodiment, the amount of residual liquid when discharging the liquid is reduced. As a result, effects such as process stabilization and a reduction in the number of cleaning cycles are obtained. [Examples]

[0122] Figure 14 illustrates a second substrate tilting mechanism related to the sample analysis device according to the present invention. Figure 14 illustrates the support base for the array plate. Figure 14(a) is a top view, where 601 is the support part, 701 is the array plate support part, 703 is the rotation shaft, 705 is the torsion spring, and 607 is the retaining part. Figure 14(b) is a cross-sectional view of section AA, and Figure 14(c) is a cross-sectional view of section BB. The operation of the liquid discharge method is the same as in Example 8. According to this embodiment, similar to Example 8, the amount of residual liquid when discharging the reagent solution is reduced. As a result, effects such as process stabilization and reduction in the number of washes are obtained. [Examples]

[0123] Figure 15 illustrates a third substrate tilting mechanism related to the sample analysis device according to the present invention. Figure 15 illustrates the support base for the array plate. The basic structure is the same as in Figure 14. Figure 15(a) is a top view. In this figure, unlike in Figure 14, the opening for supporting the array plate provided in the array plate support section 801 is rotated in plane; that is, each side of the opening is not parallel to each side of the array plate support section 801. Figure 15(b) is a top view of the array plate 100 when installed. The operation of the reagent solution discharge method is the same as in Example 6. According to this embodiment, similar to Example 6, the amount of residual liquid when discharging the reagent solution is reduced. As a result, effects such as process stabilization and reduction of the number of washes are obtained. Furthermore, when the array plate is tilted, the lowest point is created inside the frame, so the reagent solution is aspirated at the lowest point, further reducing the amount of residual solution compared to Example 6. [Examples]

[0124] Figure 16 illustrates the structure of the second frame related to the sample analyzer according to the present invention. The basic structure is the same as in Figure 10. In this figure, unlike in Figure 10, a pentagonal rubber 901 and a pentagonal frame 903 with a concave portion that contacts the rubber 901 are attached to the array plate 100. As a result, even when the array plate is tilted using the method of Example 8 or Example 9, the lowest point is created within the frame. [Examples]

[0125] The phosphorylation enzyme in a sample was detected using the method of the present invention. A glass slide was used as the substrate, and the phosphorylation substrate was immobilized as circular spots with a diameter of 1 mm. The circular spots were arranged at a pitch of 2 mm. The sample containing the phosphorylation enzyme was brought into contact with the spots. Subsequently, the phosphorylated spots were labeled with a dye (Alexa Fluor 680) to introduce the labeling substance. The observation solution was brought into contact with the first surface of the array plate containing the spots, and while the observation solution was in contact, the fluorescence generated by irradiating with semiconductor laser light at a wavelength of 670 nm was measured and imaged. The results are shown in Figure 17. In Figure 17, 800 indicates the optical information obtained in the present embodiment. 803-11 to 803- 36 The lines 802 and 900 show the optical information of multiple spots. 802 shows the background. 900 is a schematic diagram of the array plate used in this embodiment. From 903-11 9 03-36 indicates multiple spots. 901 is a microscope slide.

[0126] This embodiment includes the following methods and configurations. (Method 1) An analytical method for analyzing a sample containing a target using an array plate having multiple spots containing biological material on one side, A sample introduction step in which the sample is brought into contact with the plurality of spots and a portion of the plurality of spots is reacted with the target, A sample volume reduction step to reduce the amount of the sample that is in contact with the plurality of spots, An observation liquid introduction step, in which the surface is brought into contact with the observation liquid so that the plurality of spots come into contact with the observation liquid, The process includes an acquisition step of acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, A first labeling step of labeling the target before the sample introduction step, and An analytical method further comprising at least one of the following: a second labeling step performed after the sample volume reduction step, for labeling the spot that has reacted with the target. (Method 2) The analytical method according to Method 1, which includes the first labeling step, An analytical method comprising labeling with a fluorescent substance in the first labeling step. (Method 3) The analytical method according to Method 1, which includes the second labeling step, An analytical method comprising labeling with a fluorescent substance in the second labeling step. (Method 4) The analytical method according to method 2 or 3, wherein the acquisition step is performed by irradiating the plurality of spots with excitation light and acquiring at least one of the fluorescence light intensity information and spectral information from the plurality of spots as the optical information. (Method 5) The analysis method according to method 4, wherein the light intensity information and the spectral information are obtained in association with the addresses of the plurality of spots in the array plate. (Method 6) The array plate is transparent to the wavelength of the excitation light as described in method 4 or 5. (Method 7) The array plate is transparent to the wavelength of the fluorescence. (Method 8) The acquisition step is performed by irradiating the plurality of spots with the excitation light from the back side of one of the surfaces and detecting fluorescence from the back side, according to any one of methods 4 to 8. (Method 9) The observation solution is an analytical method according to any one of methods 1 to 8, having a refractive index of 1.40 or more and 1.46 or less. (Method 10) The aforementioned observation solution is an analytical method according to any one of methods 1 to 9, containing 40 vol% to 90 vol% of glycerol. (Method 11) The analytical method according to methods 1 to 10, wherein the second labeling step is performed by bringing at least one reagent solution into contact with the plurality of spots. (Method 12) The analytical method according to any one of methods 1 to 11, wherein the biomaterial comprises at least one of nucleic acids, peptides, and proteins. (Method 13) The aforementioned biomolecule is an analytical method according to any one of methods 1 to 11, comprising a phosphorylation enzyme substrate. (Method 14) The aforementioned reagent solution is the analytical method according to method 13, which includes a phosphorylation site recognition substance. (Method 15) The analytical method according to methods 1 to 14, further comprising a sample information acquisition step of acquiring information about the sample based on the optical information. (Method 16) The analytical method according to any one of methods 1 to 15, wherein the observation solution introduction step also serves as part of the sample volume reduction step. (Method 17) The analytical method according to any one of methods 1 to 16, wherein the sample in contact with the plurality of spots is replaced with the observation solution in contact with the plurality of spots. (Composition 1) An analytical device for analyzing a sample containing a target using an array plate having multiple spots containing biological material on one side, A sample introduction mechanism that brings the sample and the plurality of spots into contact, A sample volume reduction mechanism for reducing the amount of sample in contact with the plurality of spots, An observation liquid introduction mechanism that brings the surface into contact with the observation liquid so that the plurality of spots come into contact with the observation liquid, The system includes an acquisition mechanism that acquires optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, A first labeling mechanism that operates before the sample introduction mechanism operates and labels the target, and An analytical apparatus comprising at least one of the following: a second labeling mechanism which operates after the operation of the sample volume reduction mechanism and labels the spot that has reacted with the target. (Configuration 2) The analytical apparatus according to Configuration 1, wherein the array plate is transparent to the excitation light used in the acquisition mechanism, the acquisition mechanism irradiates the plurality of spots with the excitation light from the back side of one of the surfaces of the array plate, and acquires the optical information of the fluorescence emitted by the plurality of spots from the back side. (Composition 3) A holding mechanism that holds the observation liquid so that the plurality of spots come into contact with the observation liquid, The analytical apparatus further comprising configuration 1 or 2. (Composition 4) The analytical apparatus according to any one of configurations 1 to 3, further comprising a mechanism for tilting at least one of the array plate and the holding mechanism with respect to a horizontal plane. (Composition 5) The analytical apparatus according to configuration 4, wherein the tilting mechanism is configured to push the array plate or the holding mechanism vertically downward. (Composition 6) The analytical apparatus according to configuration 4 or 5, wherein the tilting mechanism includes either a pipette tip or a needle. (Composition 7) The analytical apparatus according to any one of configurations 1 to 6, further comprising a mechanism for acquiring information about the sample based on the optical information. (Composition 8) A mounting section on which an array plate having multiple spots containing biological material on one side is placed, A holding portion that holds the liquid so that it is in contact with one of the aforementioned surfaces, A liquid supply / drainage unit that enables the introduction of the liquid into the liquid-holding region of the holding unit, or the discharge of the liquid held by the holding unit, A moving part that moves the liquid supply and drainage part relative to the previously described mounting part, An optical system that irradiates light onto the plurality of spots and detects the light from the plurality of spots, A scanning unit that moves the optical system relative to the previously described mounting unit, An analytical apparatus comprising: a sample introduction step of bringing a sample into contact with the plurality of spots and reacting a portion of the plurality of spots with a target contained in the sample; a sample volume reduction step of reducing the amount of the sample in contact with the plurality of spots; an observation solution introduction step of bringing the surface into contact with the observation solution so that the plurality of spots are in contact with the observation solution; and an acquisition step of acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, wherein the control unit controls the time at which at least one of the following steps is performed: a first labeling step performed before the sample introduction step to label the target, and a second labeling step performed after the sample volume reduction step to label the spots that have reacted with the target. (Composition 9) The analytical apparatus according to configuration 8, wherein the control unit performs, in parallel with the execution of the acquisition step for the first array plate, at least one of the following steps for a second array plate different from the first array plate: the sample introduction step, the sample volume reduction step, the observation solution introduction step, and the first labeling step. (Composition 10) The control unit performs the sample introduction step, the sample volume reduction step, and the observation solution introduction step. The analytical apparatus according to configuration 8 or 9, which controls the above-described mounting unit, liquid supply and drainage unit, moving unit, optical system, and scanning unit in order to perform the second labeling step. (Composition 11) The control unit performs the sample introduction step, the sample volume reduction step, and the observation solution introduction step. To perform the second labeling step described above, the previously described mounting unit, the supply and drainage unit, the moving unit, the optical system, The analytical apparatus described in configuration 8 controls the scanning unit. (Composition 12) The analytical apparatus according to configuration 8 or 11, wherein the control unit performs, in parallel with the execution of the acquisition step for the first array plate, at least one of the following steps for a second array plate different from the first array plate: the sample introduction step, the sample volume reduction step, the observation solution introduction step, and the second labeling step. (Composition 13) A program that causes a computer to perform each of the steps described in any of Methods 1 through 17. [Explanation of symbols]

[0127] S1000: First marking process S2000: Second marking process S100: Sample introduction process S200: Sample volume reduction process S300: Observation solution introduction process S400: Acquisition process 100: Array Plate 101: Circuit board 103: Spot 105: Frame 107: Observation solution 143: Specimen 1001: Mounting section 1002: Liquid supply and drainage section 1003:Optical system 1004: Control System 1013: Mobile Unit 1014: Scanning Department 1015: Holding part 2000: Target 2001: Labeled substances 2002: Biomolecules 3001: Specimen Introduction Mechanism 3002: Mechanism for reducing sample volume 3003: Mechanism for introducing observation solution 3004: Acquisition mechanism 3005: First marking mechanism 3006: Second marking mechanism 4001: Analyzer 4003: Door 4005: Emergency stop switch 4051: LCD display 4053: Keyboard 4055: Mouse 4101:Work desk

Claims

1. An analytical method for analyzing a sample containing a target using an array plate having multiple spots containing biological material on one side, A sample introduction step in which the sample is brought into contact with the plurality of spots and a portion of the plurality of spots is reacted with the target, A sample volume reduction step to reduce the amount of the sample that is in contact with the plurality of spots, An observation liquid introduction step in which the surface is brought into contact with the observation liquid such that the plurality of spots come into contact with the observation liquid that replaces the liquid that was in contact with the plurality of spots in the preceding step, The process includes an acquisition step of acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, A first labeling step of labeling the target before the sample introduction step, and An analytical method further comprising at least one of the following: a second labeling step performed after the sample volume reduction step, for labeling the spot that has reacted with the target.

2. The analytical method according to claim 1, comprising the first labeling step, An analytical method comprising labeling with a fluorescent substance in the first labeling step.

3. The analytical method according to claim 1, comprising the second labeling step, An analytical method comprising labeling with a fluorescent substance in the second labeling step.

4. The analytical method according to claim 2 or 3, wherein the acquisition step is performed by irradiating the plurality of spots with excitation light and acquiring at least one of the fluorescence intensity information and spectral information from the plurality of spots as the optical information.

5. The analysis method according to claim 4, wherein the light intensity information and the spectral information are obtained in association with the addresses of the plurality of spots in the array plate.

6. The analytical method according to claim 4, wherein the array plate is transparent to the wavelength of the excitation light.

7. The analytical method according to claim 4, wherein the array plate is transparent to the wavelength of the fluorescence.

8. The analytical method according to claim 4, wherein the acquisition step is performed by irradiating the plurality of spots with the excitation light from the back side of one of the surfaces and detecting fluorescence from the back side.

9. The analytical method according to any one of claims 1 to 3, wherein the observation solution has a refractive index of 1.40 or more and 1.46 or less.

10. The analytical method according to any one of claims 1 to 3, wherein the observation solution contains glycerol in an amount of 40 vol% or more and 90 vol% or less.

11. The analytical method according to claim 1 or 3, wherein the second labeling step is performed by bringing at least one reagent solution into contact with the plurality of spots.

12. The analytical method according to any one of claims 1 to 3, wherein the biomaterial comprises at least one of nucleic acids, peptides, and proteins.

13. The analytical method according to any one of claims 1 to 3, wherein the biomaterial comprises a phosphorylation enzyme substrate.

14. The analytical method according to claim 11, wherein the reagent solution comprises a phosphorylation site recognition substance.

15. The analytical method according to any one of claims 1 to 3, further comprising a sample information acquisition step of acquiring information about the sample based on the optical information.

16. The analytical method according to any one of claims 1 to 3, wherein the observation solution introduction step also serves as part of the sample volume reduction step.

17. The analytical method according to claim 1, wherein the observation solution is a glycerol solution containing glycerol.

18. An analytical device for analyzing a sample containing a target using an array plate having multiple spots containing biological material on one side, A sample introduction mechanism that brings the sample and the plurality of spots into contact, A sample volume reduction mechanism for reducing the amount of sample in contact with the plurality of spots, An observation liquid introduction mechanism brings the surfaces of the plurality of spots into contact with the observation liquid that replaces the liquid that was in contact with the plurality of spots in the preceding step, The system includes an acquisition mechanism that acquires optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, A first labeling mechanism that operates before the sample introduction mechanism operates and labels the target, and An analytical apparatus comprising at least one of the following: a second labeling mechanism which operates after the operation of the sample volume reduction mechanism and labels the spot that has reacted with the target.

19. The analytical apparatus according to claim 18, wherein the array plate is transparent to the excitation light used in the acquisition mechanism, the acquisition mechanism irradiates the plurality of spots with the excitation light from the back side of one of the surfaces of the array plate, and acquires the optical information of the fluorescence emitted by the plurality of spots from the back side.

20. A holding mechanism that holds the observation liquid so that the plurality of spots come into contact with the observation liquid, The analytical apparatus according to claim 18, further comprising

21. The analytical apparatus according to claim 20, further comprising a mechanism for tilting at least one of the array plate and the holding mechanism with respect to a horizontal plane.

22. The analytical apparatus according to claim 21, wherein the tilting mechanism is configured to push the array plate or the holding mechanism vertically downward.

23. The analytical apparatus according to claim 22, wherein the tilting mechanism includes either a pipette tip or a needle.

24. The analytical apparatus according to any one of claims 18 to 23, further comprising a mechanism for acquiring information about the sample based on the optical information.

25. A mounting section on which an array plate having multiple spots containing biological material on one side is placed, A holding portion that holds the liquid so that it is in contact with one of the aforementioned surfaces, A liquid supply / drainage unit that enables the introduction of the liquid into the liquid-holding region of the holding unit, or the discharge of the liquid held by the holding unit, A moving part that moves the liquid supply and drainage part relative to the previously described mounting part, An optical system that irradiates light onto the plurality of spots and detects the light from the plurality of spots, A scanning unit that moves the optical system relative to the previously described mounting unit, An analytical apparatus comprising: a sample introduction step of bringing a sample into contact with the plurality of spots and reacting a portion of the plurality of spots with a target contained in the sample; a sample volume reduction step of reducing the amount of the sample in contact with the plurality of spots; an observation solution introduction step of bringing the surface of the plurality of spots into contact with an observation solution that replaces the liquid that was in contact with the plurality of spots in the preceding step; and an acquisition step of acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, wherein the control unit controls the time at which at least one of the following steps is performed: a first labeling step performed before the sample introduction step to label the target, and a second labeling step performed after the sample volume reduction step to label the spots that have reacted with the target.

26. The control unit performs the sample introduction step, the sample volume reduction step, and the observation solution introduction step. The analytical apparatus according to claim 25, which controls the above-described mounting unit, liquid supply and drainage unit, moving unit, optical system, and scanning unit in order to perform the first labeling step.

27. The analytical apparatus according to claim 26, wherein the control unit performs, in parallel with the execution of the acquisition step for the first array plate, at least one of the following steps for a second array plate different from the first array plate: the sample introduction step, the sample volume reduction step, the observation solution introduction step, and the first labeling step.

28. The control unit performs the sample introduction step, the sample volume reduction step, and the observation solution introduction step. The analytical apparatus according to claim 25, which controls the above-described mounting unit, liquid supply and drainage unit, moving unit, optical system, and scanning unit in order to perform the second labeling step.

29. The analytical apparatus according to claim 28, wherein the control unit performs, in parallel with the execution of the acquisition step for the first array plate, at least one of the following steps for a second array plate different from the first array plate: the sample introduction step, the sample volume reduction step, the observation solution introduction step, and the second labeling step.

30. A program for causing a computer to perform each of the steps described in any one of claims 1 to 3.

31. An analytical method for analyzing a sample containing a target using an array plate having multiple spots containing biological material on one side, A sample introduction step in which the sample is brought into contact with the plurality of spots and a portion of the plurality of spots is reacted with the target, A sample volume reduction step to reduce the amount of the sample that is in contact with the plurality of spots, An observation solution introduction step in which the surfaces are brought into contact with the observation solution such that the plurality of spots come into contact with the observation solution containing 40 vol% to 90 vol% of glycerol, The process includes an acquisition step of acquiring optical information of the plurality of spots while the plurality of spots are in contact with the observation solution, A first labeling step of labeling the target before the sample introduction step, and An analytical method further comprising at least one of the following: a second labeling step performed after the sample volume reduction step, for labeling the spot that has reacted with the target.

32. The analytical method according to claim 31, wherein the observation solution has a refractive index of 1.40 or more and 1.46 or less.

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