CN117957443A

CN117957443A - Specimen analysis method and specimen analysis device

Info

Publication number: CN117957443A
Application number: CN202280049548.8A
Authority: CN
Inventors: 古川幸生; 城井浩; 宇都宫纪彦; 尾嵜真浩; 佐竹皓宇
Original assignee: Canon Inc; Canon Medical Systems Corp
Current assignee: Canon Inc; Canon Medical Systems Corp
Priority date: 2021-07-13
Filing date: 2022-07-11
Publication date: 2024-04-30

Abstract

Assays using array plates typically require a drying step. However, the drying step interrupts a series of operations. In addition, uneven drying of the array plate is a factor of reducing reproducibility. The present invention proposes a method of acquiring optical information of a plurality of spots in a state where the plurality of spots are in contact with an observation liquid.

Description

Specimen analysis method and specimen analysis device

Technical Field

The present invention relates to a sample analyzer and a sample analysis method for analyzing a sample.

Background

There are known a large number of array plates such as protein arrays, peptide arrays and DNA arrays in which substances such as proteins, peptides or nucleic acids are immobilized in spots on a substrate. By using the array plate, interactions between a large amount of immobilized substances and substances contained in a specimen are observed at one time. Thus, interactions with a large number of substances including specimens derived from living bodies such as blood, cell extracts, saliva, and interstitial fluid are comprehensively analyzed.

As a measurement method using an array plate, there is known a method of selectively fluorescence-labeling a spot where an interaction of interest has occurred and acquiring optical information of the spot. In patent document 1 and patent document 2, an array plate is observed by using a microarray scanner. The apparatus of patent document 1 includes an illumination optical system, a fluorescence detection optical system, and a two-dimensional scanning system. The irradiation optical system has a function of applying the collected laser light to the array plate. The fluorescence detection optical system has a function of detecting the amount of fluorescence emitted from the fluorescence-labeled spot. The two-dimensional scanning system has a function of two-dimensionally scanning an array plate or an optical system to acquire a fluorescent image of a spot on the array plate.

Patent document 2 proposes an antibody array using a proximity vehicle for detecting oncogenic fusion proteins.

CITATION LIST

Patent literature

Patent document 1: U.S. patent application publication 2009/0218513

Patent document 2: japanese patent application laid-open No. 2013-508728

Disclosure of Invention

Technical problem

However, patent document 1 and patent document 2 have a problem in that it is necessary to observe the array plate in a dry state.

A series of operations including bringing the array plate and the specimen into contact with each other and selectively performing fluorescent labeling of spots where a reaction of interest has occurred are generally performed in a liquid. Thus, the drying step is a step of interrupting a series of operations. The drying step in the related art uses a method of physically removing a liquid in contact with a plate from the top of the plate, such as centrifugal separation or air blowing, for the purpose of reducing denaturation of biological substances and specimens, instead of using a method including a heating step using a phase change between a liquid phase and a gas phase. In the specification of the present application, the drying step is hereinafter used as a concept including a step of removing liquid from the top of the substrate by using an external force including an inertial force, which does not always require a heating step. The drying step is generally intended to remove residual components not immobilized on the spots, i.e. the next step and unwanted components thereafter, from the dissolved components of the top of the plate in reagents such as targets, enzymes and antibodies. Thus, the drying step is not intended to selectively volatilize the solvent to leave the dissolved components as a residue on top of the substrate.

Solution to the problem

Meanwhile, the inventors of the present application have visually recognized that when a drying step is performed on an array plate in contact with a reagent, as shown in fig. 18, even after drying, a trace of a part of liquid of the reagent remains to cause "drying unevenness". The formation of the liquid trace is presumed to be due to the local and long-term presence of the reagent containing the dissolved component at a higher concentration than in the other region than the liquid trace, and the liquid trace is presumed to have a larger amount of residue than in the other region.

That is, initially, a plurality of spots on an array plate need to receive a uniform history from a reaction step to an analysis step of performing analysis, but a drying step is a factor of adjusting uniformity and reproducibility of the history. FIG. 18 shows array plate 199 after drying, spots 193-11 through 193-36 on the array plate, and liquid marks 190. The liquid trace 190 means that the reaction liquid dries later than the surrounding area where the liquid trace 190 is not recognized, and there is a fear that a part of the specimen 143, the phosphorylation reaction liquid, or the dissolved component contained in the reagent partially remains as a residue as compared with the area outside the liquid trace 190. Such liquid marks 190 are marks that can be visually identified. The detection dynamic range when optically detecting information from the label is from about two digits to about five digits, and thus there is a fear that the specimen causing drying unevenness is caused to be suddenly changed into a cause of background noise in the analysis step.

In view of the above, an object of the present invention is to provide an analysis method and an analysis apparatus that enable reduction of the influence of residues caused by drying unevenness in analysis of a specimen using an array plate.

Advantageous effects of the invention

According to the present invention, it is possible to provide an analysis method and an analysis apparatus that enable reduction of the influence of residues caused by drying unevenness in analysis of a specimen using an array plate.

Drawings

Fig. 1 (a) is a diagram for illustrating the first embodiment, and fig. 1 (b) is a diagram for illustrating the second embodiment.

Fig. 2 is a view for showing a first identification step and a second identification step.

Fig. 3 is a diagram for illustrating an apparatus according to an embodiment.

Fig. 4 (a) is a view for showing a schematic configuration of the specimen analysis apparatus according to the embodiment, and fig. 4 (b) is a view for showing an arrangement on a table of the specimen analysis apparatus according to the embodiment.

Fig. 5 is a view for illustrating the process of example 1.

Fig. 6 is a view for illustrating the process of example 1.

Fig. 7 is a view for illustrating the process of example 1.

Fig. 8 is a view for illustrating the process of example 1.

Fig. 9 is a view for showing a fluorescence detection step of example 1.

Fig. 10 is a view for illustrating the structure of a frame.

Fig. 11 is a view for illustrating an optical information acquisition method.

Fig. 12 is a view for illustrating an array board support base.

Fig. 13 is a view for illustrating a reagent liquid discharging method using an array plate tilting mechanism.

Fig. 14 is a view for illustrating a second array plate tilting mechanism.

Fig. 15 is a view for illustrating a third array plate tilting mechanism.

Fig. 16 is a view for illustrating the structure of the second frame.

Fig. 17 shows the analysis results.

Fig. 18 is a view for showing a liquid trace of the array plate.

Detailed Description

According to an embodiment of the present invention, there is provided an analysis method for analyzing a specimen containing a target by using an array plate including a plurality of spots each containing a biological substance on one surface thereof, the analysis method including: a specimen introducing step of bringing the specimen and the plurality of spots into contact with each other so that a part of the plurality of spots and the target react with each other; a specimen amount reducing step of reducing an amount of a specimen in contact with the plurality of spots; an observation liquid introducing step of bringing one surface into contact with an observation liquid so that a plurality of spots are brought 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. The analysis method further includes at least any one of a first identification step of identifying the target and a second identification step of identifying a portion of the plurality of spots that react with the target before the specimen introducing step, the second identification step being performed after the specimen amount reducing step.

Furthermore, according to another embodiment of the present invention, there is provided an analysis apparatus including: a placement unit on which an array plate including a plurality of spots each containing a biological substance on one surface thereof is to be placed; a holder configured to hold a liquid such that one surface and the liquid are in contact with each other; a liquid feeder/discharger configured to introduce liquid into a liquid-holding region of the holder or discharge the liquid held by the holder; a moving unit configured to move the liquid feeder/ejector relative to the placement unit; an optical system configured to apply light to the plurality of spots to detect light emitted from the plurality of spots; a scanner configured to move the optical system relative to the placement unit; and a controller configured to control a time at which each of the following steps is performed: the method includes a specimen introducing step of bringing the specimen and the plurality of spots into contact with each other to cause a portion of the plurality of spots and a target contained in the specimen to react with each other, a specimen amount reducing step of reducing an amount of the specimen in contact with the plurality of spots, an observation liquid introducing step of bringing one surface into contact with the observation liquid so that the plurality of spots are in contact with the observation liquid, and an acquiring step of acquiring optical information of the plurality of spots in a state in which the plurality of spots are in contact with the observation liquid, the controller being further configured to control a time to perform at least any one of a first identifying step of identifying the target and a second identifying step of identifying a portion of the plurality of spots reacting with the target, the first identifying step being performed before the specimen introducing step, the second identifying step being performed after the specimen amount reducing step.

Furthermore, according to another embodiment of the present invention, there is provided an analysis apparatus including: a placement unit on which an array plate including a plurality of spots each containing a biological substance on one surface thereof is to be placed; a holder configured to hold a liquid such that one surface and the liquid are in contact with each other; a liquid feeder/discharger configured to introduce liquid into a liquid-holding region of the holder or discharge the liquid held by the holder; a moving unit configured to move the liquid feeder/ejector relative to the placement unit; an optical system configured to apply light to the plurality of spots to detect light emitted from the plurality of spots; a scanner configured to move the optical system relative to the placement unit; and a controller configured to control the placement unit, the liquid feeder/ejector, the moving unit, the optical system, and the scanner to enable execution of steps of an analysis method including: a specimen introducing step of bringing the specimen and the plurality of spots into contact with each other to cause a part of the plurality of spots and a target contained in the specimen to react with each other; a specimen amount reducing step of reducing an amount of a specimen in contact with the plurality of spots; an observation liquid introducing step of bringing one surface into contact with an observation liquid so that a plurality of spots are brought 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, the analysis method further comprising at least any one of a first identification step of identifying the target before the specimen introducing step and a second identification step of identifying a portion of the plurality of spots that react with the target, the second identification step being performed after the specimen amount reducing step.

As one embodiment, the present invention provides an analysis method for analyzing a specimen containing a target by using an array plate including a plurality of spots each containing a biological substance on one surface thereof, the analysis method comprising: a specimen introducing step of bringing the specimen and the plurality of spots into contact with each other so that a part of the plurality of spots and the target react with each other; a specimen amount reducing step of reducing an amount of a specimen in contact with the plurality of spots; an observation liquid introducing step of bringing one surface into contact with an observation liquid so that a plurality of spots are brought 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, the analysis method further comprising at least any one of a first identification step of identifying the target before the specimen introducing step and a second identification step of identifying a portion of the plurality of spots that react with the target, the second identification step being performed after the specimen amount reducing step.

< Array Board >)

The array plate includes spots containing a large number of types of biological substances on a substrate, and is used for comprehensive analysis of a sample. In some cases, the array plate is also called a microchip, a microarray, a protein chip, a DNA chip, or the like. The location of each spot in the array plate is referred to as an "address in the array plate". Each blob may be labeled with a symbol as an address. The address may be determined by the physical location of each spot on the array plate. In this case, the address may be determined by a distance or coordinates from each end of the array plate.

In the specification of the present application, the array plate includes at least sixteen spots each containing a biological substance, and further includes at least two spots different from each other in at least any one of composition, content, and amount of the biological substance. In the specification of the present application, the array plate has a flat plate shape and includes spots containing biological substances, respectively, on one surface thereof. In some cases, the surface on which the spots are disposed is also referred to as a "first surface (101-1)", and the back surface of the first surface is also referred to as a "further surface (101-2)" or a "second surface (101-2)".

The array plate in the present invention is a flat plate having a length and a width of 10mm to 128mm and a thickness of 0.1mm to 2.5mm, respectively. As the shape of the plate, a hexahedral form including a rectangular parallelepiped and a truncated rectangular pyramid or a disc form including a cylindrical body, an elliptical cylindrical body and a truncated cone is adopted. Preferably, the peripheral edge of the periphery of the surface for defining the plate is rounded or tapered. A plurality of spots are arranged on one of two surfaces opposite to each other to define the thickness of the plate. The diameter of the spot is adjusted according to the size of the plate, the image pickup field of view, and the number of spots, but a diameter of 0.1 μm to 5000 μm is adopted. As the spots, square, rectangular, circular, or elliptical arranged to be separated from adjacent spots are employed. As the period of the array between spots, a period of about 1.1 times to about 10 times the diameter of the spots is employed. An array panel comprises at least sixteen more spots, more preferably sixty-four more spots, and more preferably one thousand more spots.

Commercially available array plates may be used. Microarray plates are sold by Agilent technologies Inc. (Agilent Technologies Ltd.) and Rabo biotechnology Inc. (RayBiotech Inc.), etc. As another example, an array plate is manufactured with reference to a well-known method. An array plate is prepared by immobilizing a desired biological substance on one surface of a suitable substrate. Immobilization is also known as "adsorption", and examples thereof include immobilization caused by hydrophobic interactions, electrostatic interactions, van der waals interactions, hydrogen bonds, and covalent bonds. As the substrate, a substrate having a transmittance for the wavelength of excitation light is used. Further, a substrate having transmittance for a wavelength of fluorescence according to an optical arrangement is employed. The phrase "having transmissivity" means the property of transmitting light having at least one of the wavelengths. The phrase "having transmissivity" includes a case where a part of light having a specific wavelength is transmitted. Further, as the substrate, in some cases, a substrate which causes less scattering at the wavelength of the excitation light and the fluorescence and has transmittance is used. Examples of the material of the substrate include glass, synthetic quartz, and borosilicate glass. Examples of the material also include resins such as polystyrene, polypropylene, (meth) acrylic resin, polyamide, polyimide, melamine, ABS, polyphenoxyurethane, silicone, epoxy resin, and polydimethylsiloxane. In addition, as the material, a film such as nitrocellulose and polyvinylidene fluoride, and a gel such as agarose and acrylamide may be used. These materials may be used as an array plate by being fixed or supported by, for example, a frame-like frame or another substrate, respectively, as needed.

Immobilization of biological substances may be performed by well-known methods such as a method using a spotter (arrayer), a method performed by stamping, a method performed by using semiconductor technology, and a method by pipetting. In addition, in order to prevent substances contained in the specimen from directly binding to the substrate, the array plate may be treated with a nonspecific adsorption inhibitor containing a blocking agent.

< Biological substance >

Biological substances as used herein refer to substances immobilized on an array plate and contained in spots. The biological substance may be appropriately determined according to the purpose by the person who realizes the present invention, and any kind of substance may be a biological substance. Biological substances include, for example, proteins, peptides, nucleic acids, low molecular weight compounds, viruses and cells, and any of these substances may be naturally derived or obtained by synthesis or genetic recombination. Further, specific examples of biological substances include various substances such as antibodies, antigens, phosphorylated proteins, dephosphorylated proteins, low molecular weight compounds, receptors, enzymes, matrices, phosphorylase matrices, allergens, cytokines, hormones, bacteria, viruses, microorganisms, DNA, RNA, cDNA, cells, cell membrane constituents, cancer markers, disease markers, bio-derived substances, living extracts, and the like, blood derived substances, foods, food derived substances, natural products, natural product derived substances, and culture derived substances.

< Specimen >

The specimen may be appropriately determined according to the purpose by the person who realizes the present invention, and any kind of specimen may be the specimen. Specimens include biological derivatives, living extracts, and the like, blood derivatives, food derivatives, natural products, natural product derivatives, and culture derivatives. Substances contained in a specimen and intended to react with biological substances are referred to as targets. Examples of targets include antibodies, antigens, phosphorylated proteins, dephosphorylated proteins, nucleic acids, low molecular weight compounds, receptors, enzymes, substrates, allergens, cytokines, hormones, bacteria, viruses, microorganisms, DNA, RNA, cDNA, cells, cell membrane components, cancer markers, and disease markers. According to the method of this embodiment, information about interactions between targets and biological substances contained in a specimen can be obtained, and information about the specimen can be obtained.

Depending on the purpose and the procedure, the specimen may be suitably pretreated. A treatment such as a reduction treatment or a heat treatment may be performed. In addition, the reagent may be added to the specimen in advance. The labeling substance may be pre-bound to a target contained in the specimen.

The form of the specimen may be solid, liquid or gas, but the specimen is used in liquid form by dilution, suspension or extraction with water, physiological saline, buffer solution or other solutions as appropriate. The specimen may contain surfactants, preservatives, non-specific adsorption inhibitors or other additives. Further, the reagent is added to the specimen according to the purpose.

< Marking substance >

To visualize spots that have reacted with the target, the target or spots that have reacted with the target are identified, i.e. the target is bound to a labeling substance or the labeling substance is introduced to spots that have reacted with the target. As the labeling substance, a labeling substance capable of being optically detected is used. Examples of the labeling substance include a fluorescent substance, a chemiluminescent substance, a phosphorescent substance, a dye, gold nanoparticles, fluorescent particles, an enzyme for causing an enzymatic chemiluminescent or chromogenic reaction, and microparticles exhibiting absorption at a specific wavelength. In addition, the labeling substance may comprise antibodies, ligands or other binding sites. The binding or introduction of the labeling substance is performed by binding the labeling substance by utilizing hydrophobic interaction, electrostatic interaction, van der Waals interaction, hydrogen bond or covalent bond binding, or performing introduction at the time of PCR or synthesis. As the identification method, a known method is used.

Typically, the marking substance is a fluorescent substance. The first marking step or the second marking step, which will be described later, is generally marked with a fluorescent substance. The fluorescent substance has advantages in that luminescence can be stably obtained, a matrix is not required, and region-specific labeling is enabled, thereby being suitable for two-dimensional analysis, or multiple labeling is enabled by using fluorescent substances having different wavelengths. The kind of the fluorescent substance is appropriately selected according to the reaction system or the optical system. Fluorescent substances include, for example, fluorescein, cyanine, rhodamine, texas red, coumarin-based dyes, fluorescent proteins and quantum dots. The skilled person can choose a fluorescent substance with appropriate excitation wavelength, fluorescence wavelength and properties according to an optical system or an experimental system.

The advantage of chemiluminescent substances is that luminescence is obtained by adding a matrix and no excitation light source is required, and that the sensitivity is high. When a chemiluminescent substance is used, an enzyme or a substrate for catalyzing chemiluminescence is used as the labeling substance. Examples of substrates include enzymes such as alkaline phosphatase and horseradish peroxidase, and substrates comprising acridine. Chemiluminescence can typically be observed using a highly sensitive CCD camera. Meanwhile, a substrate or a catalyst for luminescence needs to be supplied at the time of detection. These substances are preferably added to the viewing liquid.

Gold nanoparticles have a property of absorbing light having a wavelength close to a specific wavelength according to their particle size. When the labeling substance is gold nanoparticles, the labeled spots can be detected by measuring the amount of reflected light or light absorption at selective wavelengths. Gold has a high affinity for sulfur, nitrogen and oxygen atoms, and the object to be identified is thiolated (thiolated) (adding-SH groups) to bind to the surface of the gold nanoparticle through a sulfur-gold atom bond.

< Observed liquid >

The viewing liquid is also referred to as a "wash liquid" meaning that the viewing liquid displaces, i.e., removes, the optical background noise component. The observation liquid is arranged to be in contact with at least the spots of the array plate when acquiring the optical information. The viewing liquid may be arranged in contact with a portion of or the entire first surface of the array plate. Preferably, the observation liquid has a good affinity for the liquid used on the array plate in the immediately preceding treatment. It is desirable to observe that the refractive index of the liquid is close to the refractive index of the substrate of the array plate so that oxidation of the substances on the array plate, in particular the marking substances, is prevented. Further, it is desirable that the observation liquid does not emit fluorescence when excitation light for acquiring optical information is applied thereto. The glycerin solution is suitably used as the observation liquid satisfying these conditions, but the observation liquid may be suitably selected based on the properties of the labeling substance, biological substance, and specimen.

In addition, the observation liquid may be used as the cleaning liquid. As another example, a cleaning liquid present on the array plate, a reagent to be used in the second labeling step, or any other liquid used in the process may be used as the observation liquid.

The refractive index of the observation liquid is closer to that of the substrate of the array plate than air. From the viewpoint of the refractive index being closer to that of the array plate, the observation liquid is preferably a liquid having a refractive index of 1.3 or more, more preferably a liquid having a refractive index of 1.33 to 1.60, and still more preferably a liquid having a refractive index of 1.40 or more and 1.46 or less. Examples thereof may preferably be water, physiological saline, phosphate buffer, trichloric acid buffer, any other buffer, and any other solution each containing 40vol% or more and 90vol% or less of glycerol. Glycerol has hygroscopicity and high viscosity, and when glycerol exceeds 90vol%, it is difficult to observe the use of the liquid. When the glycerin is less than 40vol%, the refractive index of the liquid is observed to be separated from the refractive index of the array plate. In addition, the observation liquid may suitably contain a surfactant, a preservative, and a nonspecific adsorption inhibitor. As the observation liquid, generally, a buffer solution containing 80vol% of glycerin can be given.

The viewing liquid may also contain commercially available anti-fade agents for preventing fading of the dye. Examples of fade resistance agents include Prolong manufactured by sameidie technologies (Thermo FISHER SCIENTIFIC inc.).

< Analytical methods >

In the analysis method of the present embodiment, part or all of the operations may be performed by a human hand, but preferably all of the steps are performed automatically by the apparatus. The method of this embodiment does not include a drying step. Thus, there is no need to transfer the array plate to a dryer, which provides the further advantage that all steps are performed automatically by the device. A conceptual diagram of steps in the present embodiment is shown in fig. 1 (a) and fig. 1 (b). Fig. 1 (a) shows an analysis method S1100 according to the first embodiment using a first identification step, and fig. 1 (b) shows an analysis method S2200 according to the second embodiment using a second identification step. The analytical method is described in detail below. However, the present invention is not limited to the following description.

First embodiment

Fig. 1 (a) shows a processing diagram of an analysis method S1100 according to the first embodiment. The analysis method S1100 includes a first identification step S1100 of identifying a target, which is performed before a specimen introducing step S100 (described later) of bringing a specimen and a plurality of spots into contact with each other to react a portion of the plurality of spots with the target with each other.

Specimen introducing step (S100)

In this step, the specimen and the spot are brought into contact with each other so that a part or all of the target reacts with the biological substance contained in the spot. Examples of reactions include binding between a target and a biological substance, chemical reactions, and enzymatic reactions.

Depending on the purpose, reagents may be added to the specimen. The specimen is added in an amount that covers at least the spots. A portion or the entire first surface of the array plate may be covered by the specimen. The substance contained in the specimen and the biological substance react with each other within a desired period of time at a temperature suitable for the reaction.

As the holding mechanism, the frame may be provided on the array plate, or the array plate may be placed in a suitable container. The specimen and the liquid used in each of the following steps, such as the observation liquid, the replacement liquid, or the reagent, may be held on the array plate by the frame or the container.

Sample amount reducing step (S200)

In this step, the amount of the specimen introduced in the specimen introducing step S100 is reduced. By this step, the target bound to the biological substance remains on the spot side in the specimen introducing step S100, and the target that does not react with the biological substance or the target obtained after the biological substance undergoes a chemical reaction or an enzymatic reaction is reduced.

The specimen on the spot is reduced by suction from the nozzle. Examples of nozzles include pipette tips, needles, pipettes, syringes, pumps, and combinations thereof. The specimen may be reduced by tilting the array plate or the container containing the array plate relative to a horizontal plane. As another example, without using a nozzle, the array plate may simply be tilted so that the specimen is reduced by decantation, and then the liquid may be further sucked by the nozzle to be reduced. The term "decreasing" here means that the volume of the liquid becomes less than 10% of the volume of the liquid before decreasing. Not only the reduction of the specimen but also the reduction of other liquids is performed similarly to the above case.

Observing the liquid introducing step (S300)

In this step, the observation liquid is brought into contact with the spots. By this step, the drying step is not required, and thus the processing from the specimen introducing step S100 to the acquiring step S400 can be performed without interruption and without leaving a drying trace.

The observation liquid is added to cover at least the spots. It is sufficient that the top of the target spot is in contact with the observation liquid, but a part or the whole of the first surface of the array plate may be covered with the observation liquid. It is preferable that the observation liquid is in contact with only the first surface side of the array plate, and the observation liquid is not in contact with the second surface.

Further, a cleaning liquid and other reagent liquids may be used as such as an observation liquid, and in this case, the step of bringing these liquids into contact with the spots corresponds to this step.

Further, the observation liquid introducing step S300 may also be used as a part of the specimen amount reducing step S200. That is, when the process of reducing a portion of the specimen on the spot, introducing the observation liquid, and reducing again a portion of the mixed liquid of these liquids is repeated a plurality of times, the amount of the specimen may be reduced to less than 10% of the introduced amount or reduced to a desired level. By this method, all steps can be performed in a state where some types of liquid are constantly kept on the spots.

Furthermore, the specimen in contact with the spot may be replaced with the viewing liquid, i.e. the introduced specimen may be replaced with the viewing liquid instead of being replaced once with another liquid.

Acquisition step (S400)

In this step, the optical information of the spot is acquired. Using the optical information, information about the presence or absence of the marker substance is obtained, i.e. it can be determined whether the spot has reacted with the target.

Optical information of a plurality of spots is obtained in a state where the spots are in contact with an observation liquid. This information is acquired by the optical system. The optical information includes at least any one of light intensity information and spectral information from the spot. The optical system is present on the second surface side of the array plate. When the labeling substance is a fluorescent substance, the optical system applies excitation light to the spot from the second surface side, and detects fluorescence emitted from the spot from the second surface side. The light intensity information and the spectral information may be acquired in association with the address of the spot in the array plate. Information about the specimen may be further acquired based on the obtained optical information. This step may be referred to as a "specimen information acquisition step".

The refractive index of the observation liquid is closer to that of the array plate than air, and therefore light emitted from the optical system is less likely to be reflected at the interface between the array plate and the observation liquid. Thus, optical information with a higher signal-to-noise ratio can be obtained.

When the labeling substance is a fluorescent substance, any optical system may be used without limitation as long as the optical system can excite the fluorescent substance and detect fluorescence emitted from the spot. For exciting the fluorescent substance, an excitation light source is used, and, for example, a laser light source, a light emitting diode, an LED, a mercury arc, and a halogen tungsten lamp can be used as the excitation light source. For example, a CCD camera or photodiode may be used for detection. The optical system suitably comprises a filter for illuminating or detecting light having a defined wavelength. In addition, the optical system may include a lens. The optical system may be of a scanning type or a non-scanning type. A typical optical system is a confocal optical unit.

When, for example, chemiluminescent, gold colloids or dyes are used as the marking substance, no excitation light source is required.

First identification step (S1000)

In the analysis method S1100 according to the first embodiment and the analysis method S2200 according to the second embodiment described later, at least any one of the first identification step S1000 and the second identification step S2000 is performed in order to identify a blob. In the case where the analysis is performed by binding the labeling substance to the target contained in the specimen in advance, the first labeling step S1000 is performed. When the target bound to the labeling substance is bound to the biological substance in the specimen introducing step S100, the spot having the bound target can be observed in a labeled manner in the acquiring step S400, and thus it can be determined that the reaction has occurred.

Examples of methods of binding the labeling substance to the target include chemical reaction via a functional group, methods using PCR, methods using a kit, and any other well-known methods. This step is performed before the specimen is introduced into step S100. In fig. 2 (a), an outline of the case where the first identification step S1000 is performed is shown. In a first identification step S1000, the target 2000 is identified with the identification substance 2001 (the uppermost view and the second view of fig. 2 (a)). In the specimen introducing step S100 and the specimen amount reducing step S200, the target reacts with the biological substance 2002 contained in the spot 103, and the marker substance 2001 is introduced to the spot 103 (the third view and the lowermost view of fig. 2 (a)). In the observation liquid introducing step S300, the spot and the observation liquid are brought into contact with each other.

Second embodiment

Fig. 1 (b) shows a processing diagram of an analysis method S2200 according to the second embodiment. The analysis method S2200 is different from the first embodiment S1100 in that it includes a second identification step S2000 of identifying a target, which is performed before the specimen introducing step S200 of reducing the amount of the specimen in contact with the plurality of spots.

Second identification step (S2000)

When the first identification step S1000 is not performed, the second identification step S2000 is performed. Both the first identification step S2000 and the second identification step S2000 may be performed. The second identifying step S2000 is performed after the specimen-amount reducing step S200, and identifies spots that have reacted with the target. The marker substance is specifically introduced to the target bound to the spot or to a change in biological substance caused by the target. In this step, the spots that react with the target can be observed in a labeled manner, and thus it can be determined that a reaction has occurred.

Fig. 2 (b) shows an outline of the case where the second identification step S2000 is performed. The target 2000 reacts with the spot 103 through the specimen introducing step S100 (uppermost view of fig. 2 (b)) and the specimen amount reducing step S200 (second view from the top of fig. 2 (b)), thereby causing a change 2003 specific to the biological substance 2002 of the spot 103. In the second labeling step S2000, the labeling substance 2001 is specifically introduced to the variation 2003 of the biological substance (third view from the top of fig. 2 (b)). An example of a change 2003 in biological material is phosphorylation. The labeling substance is introduced via an antibody, ligand or a second antibody thereof that recognizes a change in biological substance (the lowermost view of fig. 2 (b)). Other known methods may be used in this step. In the second labeling step S2000, the labeling substance is not introduced (immobilized) to unreacted spots that do not react with the target, but in this case, the labeling substance exists in a non-immobilized state on the substrate between the unreacted spots. Thus, the marking substance becomes background noise in the acquisition step. The blobs are identified by either or both of the first identification step S1000 and the second identification step S2000.

To complete the second marking step S2000 to achieve a state in which the marking substance is introduced into a portion of the plurality of spots, a cleaning step is performed to reduce the non-immobilized (non-introduced) marking substance present on the substrate between the unreacted spots from the first surface. In the cleaning step, the reagent liquid 125-2 containing the labeling substance 2001 is reduced from one surface (the first surface 101-1) of the substrate, so that the non-immobilized labeling substance present on the substrate between the unreacted spots is reduced. In other words, the cleaning step reduces the non-immobilized marking substances present on the substrate between the unreacted spots, such that the marking substances introduced to the spots that have reacted with the target are selectively left in the respective spots. In some cases, the cleaning step described later is also referred to as a part of the second identification step S2000.

The residues of the reagent liquid 125-2 used in the second identification step S2000 on the first surface 101-1 are replaced with a cleaning liquid for cleaning in each cleaning step. Thus, the cleaning liquid is also referred to as "replacement liquid for reagent liquid 125-2". By replacing the cleaning liquid multiple times, the residue of the reagent liquid 125-1 on the first surface 101-1 is asymptotically reduced to zero. As described later, in an embodiment using a cleaning liquid as the observation liquid, the observation liquid is substantially a replacement liquid for the replacement reagent liquid 125-2. Bringing the predetermined component closer to 0 by liquid replacement is also referred to as "purge" or "cleaning", and thus the observation liquid and the cleaning liquid are also referred to as "cleaning liquid".

Cleaning step

Embodiments may include a cleaning step. The cleaning step is performed by disposing a cleaning liquid on the spots and reducing the solution after cleaning. The cleaning step may be repeated. A cleaning liquid obtained by appropriately adding a surfactant, a preservative, a non-specific adsorption inhibitor, glycerin, or any other additive to water, physiological saline, phosphate buffer, trichloric acid buffer, any other buffer, or any other solution is used as the cleaning liquid. TBST used as a ternary buffer containing polysorbate can be generally used as the cleaning liquid. The cleaning liquid may be used as such as an observation liquid. That is, a cleaning step may be included as a part of the observation liquid introducing step S300 described later.

< Detection of protein-protein interaction >

As an example, observations of protein-protein interactions may be given. As an example thereof, a method of detecting antibodies contained in a specimen by using an array plate on which an antigen is immobilized is described below.

When detecting antibodies contained in a specimen, an antigen is used as a biological substance of an array plate. The fluorescent substance is bound to the antibody contained in the specimen by the first labeling step S1000. By the specimen introducing step S100 described above, among the antibodies contained in the specimen, the antibodies that exhibit specific interactions with the predetermined antigen (biological substance) contained in the corresponding spot are bound to the antigen of the corresponding spot and immobilized on the predetermined spot. In some cases, a cleaning step is performed between the steps in the specimen amount reducing step S200, the observation liquid introducing step S300, and the acquiring step S400.

As another example, the specimen introducing step S100 and the specimen amount reducing step S200 may be performed without performing the first identifying step S1000.

In this case, the second identification step S2000 is performed. In a second labeling step S2000, a labeling substance is introduced to the targets bound to the spots via a more specific substance. Both the first identification step S1000 and the second identification step S2000 may be performed. Then, the observation liquid introducing step S300 and the acquiring step S400 are performed. The cleaning step is suitably performed between the steps. The cleaning liquid in the cleaning step may be used as the observation liquid.

Next, a method of detecting an antigen contained in a specimen by using an array plate on which an antibody is immobilized is described below. In this case, after the specimen introducing step S100 and the specimen amount reducing step S200 are performed, the identification substance is introduced in the second identification step S2000. In the specimen introducing step S100, the antigen contained in the specimen is bound to the antibody immobilized on the corresponding spot to be immobilized on the corresponding spot. Herein, the corresponding spots refer to spots including antibodies that specifically interact with antigens contained in a specimen as biological substances. In the second labeling step S2000, a labeling substance may be introduced by using an antibody recognizing an antigen. The antibody used at this time may be the same antibody as that used as the biological substance, or may be an antibody having a recognition site different from that of the antibody used as the biological substance. In the specification of the present application, in some cases, basic steps of each treatment are described while focusing on spots that cause specific interactions with targets contained in specimens, antibodies contained in reagents, or reaction promoting components. In some cases, the eye spot is referred to as a "corresponding spot". Furthermore, in some cases, a spot comprising a biological substance that specifically interacts with a target contained in a specimen is referred to as a "corresponding spot".

< Detection of nucleic acid >

As one example, detection of nucleic acids contained in a specimen is given. When detecting nucleic acids contained in a specimen, the biological substance of the array plate is synthetic oligo-DNA. Through the first labeling step S1000, a labeling substance is bound to nucleic acids contained in a specimen. The synthetic oligo-DNA has a sequence complementary to the desired DNA sequence. In the specimen introducing step S100, a hybridization reaction is performed. The specimen amount reduction step S200, the observation liquid introduction step S300, and the acquisition step S400 are performed. The cleaning step is performed as needed. The cleaning liquid in the cleaning step may be used as the observation liquid.

< Detection of phosphorylase >

As one example, the detection of phosphorylase contained in a specimen is given. The method for detecting phosphorylase contained in a specimen is described below.

When phosphorylase is detected, the biological substance of the array plate is a phosphorylase substrate. Phosphorylase matrix refers to a matrix that is phosphorylated by phosphorylase and is a generic term for proteins or peptides. In the specimen introducing step S100, the matrix of the corresponding spot is phosphorylated by a phosphorylase contained in the specimen. Here, the corresponding spots refer to spots comprising, as biological substances, a phosphorylase substrate subjected to specific phosphorylation reactions by phosphorylases contained in a specimen. After the specimen amount reducing step S200, a second identifying step S2000 is performed. In a second identification step S2000, the identification substance is introduced to the corresponding spot via the phosphorylation site-identifying substance. The phosphorylating site-recognizing substance refers to a substance that specifically recognizes a phosphorylated site in a substrate that is phosphorylated by a phosphorylase. As the phosphorylation site-recognizing substance, an anti-phosphorylated amino acid antibody is exemplified. When the labeling substance is not bound to the phosphorylation site-recognizing substance, the labeling substance is introduced via a secondary antibody or a specific reaction including biotin-avidin reaction. Then, the observation liquid introducing step S300 and the acquiring step S400 are performed. The cleaning step is performed as needed. The cleaning liquid in the cleaning step may be used as the observation liquid. The reagent liquid used in the second labeling step S2000 contains at least a primary antibody including a phosphorylation site recognition substance that recognizes the site to be phosphorylated, and if necessary, a secondary antibody including a labeling substance.

< Device >

Further, as one embodiment, the present invention provides an analysis device for analyzing a specimen containing a target by using an array plate including a plurality of spots each containing a biological substance on one surface thereof, the analysis device comprising: a specimen introducing mechanism configured to bring a specimen and a plurality of spots into contact with each other; a specimen amount reduction mechanism configured to reduce an amount of a specimen in contact with the plurality of spots; an observation liquid introducing mechanism configured to bring the one surface into contact with an observation liquid so that a plurality of spots are brought into contact with the observation liquid; and an acquisition mechanism configured to acquire optical information of the plurality of spots in a state in which the plurality of spots are in contact with the observation liquid, the analysis apparatus further comprising at least any one of a first identification mechanism configured to operate before the specimen introducing mechanism operates and identify the target, and a second identification mechanism configured to operate after the specimen amount reducing mechanism operates and identify a portion of the plurality of spots that react with the target.

The apparatus according to the present embodiment is described with reference to fig. 3 (a). The apparatus includes a specimen introducing mechanism 3001, a specimen amount reducing mechanism 3002, an observation liquid introducing mechanism 3003, and an acquisition mechanism 3004, and further includes at least any one of a first identification mechanism 3005 and a second identification mechanism 3006.

The specimen introducing mechanism 3001, the specimen amount reducing mechanism 3002, and the observation liquid introducing mechanism 3003 are mechanisms for performing the steps described above as the specimen introducing step S100, the specimen amount reducing step S200, and the observation liquid introducing step S300, respectively. The first identification mechanism 3005 and the second identification mechanism 3006 are mechanisms for performing the steps described above as the first identification step and the second identification step, respectively.

The device may further comprise a holding mechanism for holding a liquid including the observation liquid on the array plate. The holding mechanism may be included in the array plate or may be provided separately from the array plate. As the holding mechanism, a frame provided on the array plate may be given, or a container in which the array plate can be stored may be given.

Furthermore, to facilitate the pumping of liquid from the holding means, the device may further comprise means for tilting the array plate or the holding means relative to the horizontal plane. The mechanism for tilting may be configured to press the array plate or the holding mechanism vertically downward. The array plate may be tilted relative to the horizontal when the array plate or the holding mechanism is pressed by the distal end of the nozzle. Specifically, at least any one of the pipette tip or the needle may be used to press the array plate and the holding mechanism. As a mechanism for tilting, an elastic member such as a plate spring or a torsion spring may be included in a base for supporting the array plate or the holding mechanism.

To facilitate the suction of the liquid, the holding mechanism may be set to have a lowest point. The phrase "having the lowest point" means that when the holding mechanism is tilted by the above mechanism, the holding mechanism is set such that one of its vertices or one of its parts reaches a position lower than the other points. With such a design, when the holding mechanism is tilted, the liquid is collected to a point to be easily sucked. The holding mechanism is set to have the lowest point, and therefore the bottom surface of the mechanism for holding the observation liquid can be set to have any shape in consideration of the ease of suction of the liquid. For example, the shape of the bottom surface may be not only a general quadrangle, but also a triangle, pentagon, other polygon, sector, circle, ellipse, or any other shape.

Further, as still another embodiment, the present invention provides an analysis apparatus including: a placement unit on which an array plate including a plurality of spots each containing a biological substance on one surface thereof is to be placed; a holder configured to hold a liquid such that the one surface and the liquid are in contact with each other; a liquid feeder/discharger configured to introduce liquid into a liquid-holding region of the holder or discharge the liquid held by the holder; a moving unit configured to move the liquid feeder/ejector relative to the placement unit; an optical system configured to apply light to the plurality of spots to detect light emitted from the plurality of spots; a scanner configured to move the optical system relative to the placement unit; and a controller configured to control a time at which each of the following steps is performed: the method includes a specimen introducing step of bringing the specimen and the plurality of spots into contact with each other to cause a portion of the plurality of spots and a target contained in the specimen to react with each other, a specimen amount reducing step of reducing an amount of the specimen in contact with the plurality of spots, an observation liquid introducing step of bringing the one surface into contact with the observation liquid so that the plurality of spots are in contact with the observation liquid, and an acquiring step of acquiring optical information of the plurality of spots in a state in which the plurality of spots are in contact with the observation liquid, the controller being further configured to control a time to perform at least any one of a first identifying step of identifying the target, which is performed before the specimen introducing step, and a second identifying step of identifying a portion of the plurality of spots reacting with the target, which is performed after the specimen amount reducing step.

In the present embodiment, the controller may control the placement unit, the liquid feeder/ejector, the movement unit, the optical system, and the scanner to perform the specimen introducing step S100, the specimen amount reducing step S200, the observation liquid introducing step S300, and the first identifying step S1000.

At this time, the controller may perform at least any one of the specimen introducing step S100, the specimen amount reducing step S200, the observation liquid introducing step S300, and the first identifying step S1000 for a second array plate different from the first array plate in parallel with the execution of the acquiring step for the first array plate.

Further, in the present embodiment, the controller may control the placement unit, the liquid feeder/ejector, the moving unit, the optical system, and the scanner to perform the specimen introducing step S100, the specimen amount reducing step S200, the observation liquid introducing step S300, and the second identifying step S2000.

At this time, the controller may perform at least any one of the specimen introducing step S100, the specimen amount reducing step S200, the observation liquid introducing step S300, and the second identifying step S2000 for a second array plate different from the first array plate in parallel with the execution of the acquiring step for the first array plate.

An analysis device according to the present embodiment is described with reference to fig. 3 (b). The placement unit 1001 is a unit on which an array board is to be placed. The array plate includes a plurality of spots each containing a biological substance on a first surface thereof. The holder 1015 holds the liquid such that the liquid is in contact with at least the first surface of the array plate. The holder 1015 may be included in the array plate, may be provided separately from the array plate, or may be provided in the placement unit 1001.

The liquid feeder/discharger 1002 introduces a predetermined liquid into the holder 1015 or discharges the liquid held by the holder 1015. The liquid feeder/ejector 1002 may include a nozzle capable of sucking and ejecting liquid, and may further include a specimen bottle for storing a specimen, a reagent liquid bottle for storing a reagent liquid, and an observation liquid bottle for storing an observation liquid. Nozzles are a generic term for devices for sucking and discharging liquids. Examples of nozzles include pipette tips, needles, pipettes, syringes, pumps, and combinations thereof. However, when the liquid is discharged as a waste liquid, in order to prevent the waste liquid from flowing back, it is preferable to use a vacuum pump in combination so as to keep the waste liquid bottle at a negative pressure. When a vacuum pump is used, the waste liquid is discharged as needed by using a solenoid valve line.

The moving unit 1013 moves the liquid feeder/ejector 1002 relative to the placement unit 1001. An example of the moving unit is a stage for moving the nozzle.

The optical system 1003 applies excitation light to spots of the array plate 100 on the placement unit 1001, and detects the generated fluorescence. When the area that can be observed by the optical system 1003 is limited, the scanner 1014 scans the optical system 1003, the placement unit 1001, or a part of the optical system 1003 or the placement unit 1001 in order to observe optical information of the entire required area of the array board. The scanner 1014 may scan the placement unit 1001, or scan the optical system 1003. As another example, the scanner 1014 may scan only a portion of the optical system 1003 and scan only the excitation light source. In the case where the optical system 1003 exists on the second surface 101-2 side of the array plate 100 different from the first surface 101-1, interference between the optical system 1003 and the nozzles included in the liquid feeder/discharger 1002 can be avoided. Furthermore, the space can be effectively utilized, and the size of the entire apparatus can be advantageously reduced.

The phrase "moving relative to the placement unit 1001" refers to a movement performed to introduce liquid into or discharge liquid from an array plate placed on the placement unit 1001 or to decrease the distance between the liquid feeder/discharger 1002 or the optical system 1003 and the placement unit or increase the distance between the liquid feeder/discharger 1002 or the optical system 1003 and the placement unit 1001 in order to acquire optical information of spots of the array plate placed on the placement unit 1001.

The control system 1004 controls at least the execution sequence of steps related to the constituent elements of the analysis device 4001. Constituent elements of the analysis device 4001 include a placement unit 1001, a liquid feeder/ejector 1002, a moving unit 1013, an optical system 1003, and a scanner 1014.

Further, the control system 1004 controls at least the execution order of steps related to constituent elements of the analysis method. The analysis method is an analysis method including a first identification step, specifically including a first identification step S1000, a specimen introduction step S100, a specimen amount reduction step S200, an observation liquid introduction step S300, and an acquisition step S400 as shown in fig. 1 (a), and is an analysis method including a second identification step S2000, specifically including a specimen introduction step S100, a specimen amount reduction step S200, a second identification step S2000, an observation liquid introduction step S300, and an acquisition step S400 as shown in fig. 1 (b). The analysis method need only include at least either one of the first identification step S1000 and the second identification step S2000, but the analysis method may include both identification steps. Thus, in some cases, the control system 1004 is also referred to as a "sequencer" or "controller. In other words, the control system 1004 controls at least the time of executing each of the steps related to the constituent elements of the analysis method, and the control system 1004 controls at least the time of executing each of the steps related to the constituent elements of the analysis method.

The control system 1004 may control the placing unit, the liquid feeder/ejector, the moving unit, the optical system, and the scanner to perform the specimen introducing step S100, the specimen amount reducing step S200, the observing liquid introducing step S300, and the first identifying step S1000.

At this time, the controller may perform at least any one of the specimen introducing step S100, the specimen amount reducing step S200, the observation liquid introducing step S300, and the first identifying step S1000 for a second array plate different from the first array plate in parallel with the execution of the acquiring step S400 for the first array plate.

Further, in the present embodiment, the control system 1004 may control the placement unit 1001, the liquid feeder/ejector 1002, the moving unit 1013, the optical system 1003, and the scanner 1014 to perform the specimen introducing step S100, the specimen amount reducing step S200, the observing liquid introducing step S300, and the second identifying step S2000.

The control system 1004 has a function of a computer. The control system 1004 may be integrally formed with a desktop Personal Computer (PC), a laptop PC, a tablet PC, a smart phone, or the like. The control system 1004 controls the placement unit 1001, the liquid feeder/ejector 1002, the moving unit 1013, the optical system 1003, and the scanner 1014 so that the steps of the analysis method can be performed. The analysis method comprises the following steps: a specimen introducing step of bringing a specimen and a plurality of spots into contact with each other to react a part of the plurality of spots with a target; a specimen amount reducing step S200 of reducing the amount of the specimen in contact with the plurality of spots; an observation liquid introducing step S300 of bringing the first surface of the array plate into contact with the observation liquid so that the plurality of spots are brought 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. The analysis method further comprises at least any one of the first identification step S1000 and the second identification step S2000. The first identification step S1000 is a step of identifying a target before the specimen introduction step. The second labeling step S2000 is a step of labeling spots reacting with the target, which is performed after the specimen-amount reduction step S200.

In order to realize a function as a computer configured to perform arithmetic operations and storage, the control system 1004 includes a CPU 1005, a RAM1006, a ROM 1007, and an HDD 1008.CPU, RAM, ROM and HDD represent central processing units, random access memories, read only memories and hard disk drives, respectively. Further, the control system 1004 includes a communication interface (I/F) 1009, a display device 1010, and an input device 1011. The CPU 1005, RAM1006, ROM 1007, HDD 1008, communication I/F1009, display device 1010, and input device 1011 are connected to each other via a bus 1012. The display device 1010 and the input device 1011 may be connected to the bus 1012 via a driving device (not shown) for driving these devices.

In fig. 3 (b), various components forming the control system 1004 are shown as integrated devices, but part of the functionality of these components may be implemented by external devices. The display device 1010 and the input device 1011 may be external devices different from components implementing the functions of the computer including the CPU 1005.

The CPU 1005 is configured to perform predetermined operations according to programs stored in, for example, the ROM 1006 and the HDD 1008, and also has a function of controlling the respective components of the control system 1004.

The RAM 1006 is constructed from a volatile storage medium and is configured to provide a temporary memory area required for the operation of the CPU 1005. The ROM 1007 is constructed of a nonvolatile storage medium and is configured to store information required for a program for controlling the operation of the system 1004. The CPU 1005 loads a program stored in the ROM 1007 to the RAM 1006 to execute the program, thereby realizing the functions of the placement unit 1001, the liquid feeder/ejector 1002, and the optical system 1003. The HDD 1008 is formed of a nonvolatile storage medium, and is a storage device for storing information on the number of spots of an array plate and the positions thereof, as well as light intensity information or spectrum information.

The communication I/F1009 is a communication interface based on standards including Wi-Fi (trademark) and 4G, and is a module for communicating with another device. The display device 1010 is, for example, a liquid crystal display or an Organic Light Emitting Diode (OLED) display, and is used to display, for example, moving images, still images, and characters. The input device 1011 is, for example, a button, a touch pad, a keyboard, or a pointing device, and is used by the user to operate the control system 1004. The display device 1010 and the input device 1011 may be integrally formed as a touch panel.

In addition to the configuration shown in fig. 3 (b), devices may be added, and some devices may be omitted. In addition, some devices may be replaced by other devices having similar functions. In addition, some of the functions may be provided by another device via a network, and furthermore, the functions constituting the present embodiment may be implemented in a manner distributed among a plurality of devices. The HDD 1008 may be replaced with a Solid State Drive (SSD) using semiconductor elements including flash memory, or may be replaced with cloud storage.

The control system 1004 may control the operation of the optical system 1003 and the operation of the liquid feeder/ejector 1002 and the like to be performed in parallel with each other. That is, in parallel with the execution of the acquisition step of the first array plate, the control system 1004 may execute and control steps including at least any one of the specimen introducing step, the specimen amount reducing step S200, the observation liquid introducing step S300, the first identifying step S1000, and the second identifying step S2000 for the second array plate. In this way, analysis can be performed simultaneously on a plurality of array plates, thereby saving analysis time.

< Procedure >

As one embodiment, the present invention provides a program for causing a control system in an analysis apparatus to execute the series of controls described above. Further, the present invention provides a program for causing a computer to execute the above method.

Embodiments of the present invention will now be described with reference to the accompanying drawings. The present invention is not limited to the following description.

With reference to fig. 4 (a) and 4 (b), the analysis device 4001 according to the present embodiment and the arrangement of the analysis device 4001 on a table are described.

Fig. 4 (a) shows a substrate 101 and spots 103, which are embodied as slides. One type of biological substance is immobilized on each spot. The array plate 100 includes a substrate 101 and spots 103. A frame 105 serving as a holding mechanism is provided on the array board 100, and is one example of a holder 1015. The viewing liquid 107 is held inside the frame 105.

The pipette operation unit 117 is mounted to the vertical stage 113 through the intermediary of the pipette support section 115. The vertical stage 113 is mounted to the horizontal stage 11. The vertical stage 113 is movable in the vertical direction, and the horizontal stage 111 is movable in the horizontal direction. Thus, the pipette support section 115, the pipette operation unit 117, the pipette tip mounting/removing section 119, and the pipette tip 151-1 can move in the vertical direction and the horizontal direction.

The pipette operation unit 117 and the pipette tip mounting/removing section 119 are examples of a liquid feeder/ejector 1002, respectively, and the vertical stage 113 and the horizontal stage 11 are examples of a moving unit 1013, respectively.

A pipette tip holder 121 is provided, and a state in which unused pipette tips (151-2, 151-3, …) are arranged is shown.

Reagent liquid bottles 123 (123-1, 123-2) hold reagent liquid, and observation liquid bottles 124 hold observation liquid. Reagent liquid 125 (125-1, 125-2) and observation liquid 107 are provided. Reagent liquid refers to a liquid to be added to a specimen. In the present embodiment, examples of the reagent liquid include a reaction liquid, a reaction stopping liquid, a cleaning liquid, and a labeling substance.

A specimen bottle 141 and a specimen 143 are provided. A waste liquid bottle 127 and a waste liquid 153 are provided. A waste tip box 129 and a waste tip 155 are provided.

In fig. 4 (a), two reagent liquid bottles are drawn for convenience, but in actual cases, an appropriate number of reagent liquid bottles may be prepared as needed. Also for convenience, three pipette tips are drawn, but in actual practice, a required number of pipette tips are prepared as needed.

A confocal optical unit 221 is provided as an example of the optical system 1003. The optical unit 221 is mounted to the horizontal stage 223, and is movable in the horizontal direction. The horizontal stage 223 is an example of a scanner 1014.

In this embodiment, an example in which a fluorescent substance is used as a labeling substance is described.

The optical unit 221 exists at the second surface side of the array plate 100.

The components forming the optical unit 221 are described below. The optical unit 221 includes a semiconductor laser 201 for emitting light having a wavelength of 670nm, a collimator lens 203, a band-pass filter 205 for transmitting light having a wavelength of about 670nm, a long-pass filter 207 having a cut-off wavelength of 685nm, and an objective lens 209. Light emitted from the semiconductor laser passes through the collimator lens 203 and the band-pass filter 205, is reflected by the long-pass filter 207, and is collected by the objective lens 209 to the spot 103 on the first surface 101-1 of the array plate 100.

The optical unit 221 further includes a band-pass filter 211 for transmitting light having a wavelength of about 716nm, an imaging lens 213, a pinhole 215, and a photomultiplier tube 217. The light emitted from the spot passes through the objective lens 209 and the long-pass filter 207, and is collected by the imaging lens 213. Light that has passed through the pinhole 215 is detected by a photomultiplier tube 217.

When the optical unit 221 is scanned along the array plate 100 by the horizontal stage 223, two-dimensional images of a plurality of spots of the array plate 100 are acquired.

According to the present embodiment, the optical information of the spot 103 can be obtained in a state where the observation liquid 107 is held by the holding mechanism provided on the array plate 100 and the spot is in contact with the observation liquid.

Fig. 4 (b) shows a mode in which the analysis device according to the present embodiment is arranged on a table.

Fig. 4 (b) shows a sample analyzer 4001. The analysis device 4001 is arranged on the work table 4101. The analysis device 4001 according to the present embodiment includes a door 4003 for opening and closing a port for inserting and removing a reagent into and from the device. An interlock mechanism for stopping the stage for dispensing or for stopping the light emission of the laser light source may be provided when the door 4003 is opened during operation of the analyzing apparatus 4001. The analysis device 4001 includes an emergency stop switch 4005 that is activated when the operator identifies other anomalies.

The liquid crystal display 4051, the keyboard 4053, and the mouse 4055 serving as display devices correspond to an input unit for giving an instruction of a measurement condition and a higher-order instruction about a controller (not shown) from an operator. The controller may be in a mode built into the analysis apparatus 4001, or in a mode set in a computer of a local PC or on the cloud. The local PC may be disposed on or below the work table 4101. The controller may control the time at which each of the specimen introducing step S100, the specimen amount reducing step S200, the observation liquid introducing step S300, and the obtaining step is performed. Further, according to the present embodiment, specimen analysis can be performed in an environment having good workability.

Example 1

An analytical method according to the invention is described. Here, a case is exemplified in which a cell extract is used as a specimen for the purpose of analyzing a phosphorylation pathway, and a target is a phosphorylase. The array plate includes spots each containing a protein as a biological substance, the protein being a substrate in which a phosphorylation reaction is specifically catalyzed by a phosphorylase substrate. Fluorescent substances are used as the labeling substance.

Fig. 5 to 7 are views for illustrating the processing steps in example 1.

(1) Preparation (FIG. 5 (a))

The array plate 100 is arranged at a predetermined position, in the array plate 100, spots 103 on which a plurality of types of proteins are immobilized respectively on a substrate 101 are arranged in an array, and a frame 105 serving as a holding mechanism is provided around the spots 103.

The pipette tip holder 121 including the unused pipette tips 151 (151-1, 151-2, 151-3 …) is arranged at a predetermined position. For convenience, three pipette tips are shown in fig. 5 (a).

The reagent liquid bottle 123 (123-1, 123-2, … …) holding the reagent liquid 125 (125-1, 125-2, …) required for the reaction and the observation liquid bottle 124 holding the observation liquid 107 are arranged at predetermined positions. Although two reagent liquid bottles are shown in fig. 5 (a) for convenience, in actual practice, a required number of reagent liquid bottles are used.

In this example, the reagent liquid 125-1 contains a phosphorylation promoting ingredient 125-10. The reagent liquid 125-2 is a liquid containing a fluorescent dye (Alexa flow 680) conjugated secondary antibody as a fluorescent substance. The fluorescent dye conjugated secondary antibody corresponds to the labeling substance 2001. The observation liquid 107 is a glycerin solution. In addition, although not shown, in some cases, the chemical liquid container is set so that a desired solution, such as a phosphorylation reaction stopping liquid, TBST, a primary antibody liquid, a discoloration inhibitor, or pure water, can be supplied.

Further, a specimen bottle 141 containing a specimen 143 to be analyzed is arranged at a predetermined position. The specimen 143 contains a target 2000. In this example, the specimens were obtained by lysing model cells of human origin after culturing.

An empty waste bottle 127 and an empty waste tip tank 129 are installed.

(2) Mounting of pipette tip (FIG. 5 (b))

The horizontal stage 111 and the vertical stage 113 are controlled so that the pipette tip 151-1 is mounted to the pipette tip mounting/removing section 119.

(3) Suction of specimen (FIG. 5 (c))

The horizontal stage 111 and the vertical stage 113 are controlled so that the pipette tip 151-1 moves into the specimen bottle 141, and the pipette operation unit 117 is controlled so that the specimen 143 is sucked.

(4) Mixing of sample with phosphorylation reaction liquid (FIG. 6 (d))

The pipette operation unit 117 is controlled so that the specimen 143 is sucked and discharged to the reagent liquid bottle 123-1. The reagent liquid 145 is obtained by mixing the specimen 143 with the reagent liquid 125-1 containing the phosphorylation promoting ingredient 125-10. The pipette tip 151-1 is used for stirring when the reagent liquid and the specimen are mixed with each other, as needed. The reaction between the specimen and the phosphorylation reaction-promoting component may take place over time after mixing, and thus it is preferred to perform mixing immediately before the mixture is brought into contact with the spot.

(5) Suction of reagent liquid (FIG. 6 (e))

The horizontal stage 111 and the vertical stage 113 are controlled so that the pipette tip 151-1 moves into the reagent liquid bottle 123-1, and the pipette operation unit 117 is controlled so that the reagent liquid 145 is sucked.

(6) Supply of reagent liquid (FIG. 6 (f))

The horizontal stage 111 and the vertical stage 113 are controlled so that the pipette tip 151-1 moves to a position above the array plate 100, and the pipette operation unit 117 is controlled so that the reagent liquid 145 is supplied onto the spot 103.

(7) Phosphorylation reaction (not shown)

The array plate 100 is left to stand for about 5 hours so that the phosphorylation reaction is promoted.

(8) Reduction of reagent liquid (FIG. 7 (g))

The horizontal stage 111 and the vertical stage 113 are controlled such that the distal end of the pipette tip 151-1 is moved to a position directly above the array plate 100, and the pipette operation unit 117 is controlled such that the reagent liquid 145 is sucked.

(9) Discarding of pipette tip (FIG. 7 (h))

The horizontal stage 111 and the vertical stage 113 are controlled so that the pipette tip 151-1 moves to a position above the waste liquid bottle 127, and the pipette operation unit 117 is controlled so that the reagent liquid 145 is discharged. Then, the pipette tip 151-1 is moved to a position above the waste tip box 129 to be discarded.

(10) Stop of the phosphorylation reaction (not shown)

By a method similar to items (2), (5), (6), (8) and (9), the supply and reduction of the liquid are stopped by performing the phosphorylation reaction.

(11) Cleaning (not shown)

For cleaning, the supply and reduction of TBST are performed using methods similar to items (2), (5), (6), (8) and (9).

(12) Introduction of primary antibodies (not shown)

Using a method similar to items (2), (5), (6), (8) and (9), the primary antibody liquid was supplied, allowed to stand for one hour, and the primary antibody liquid was reduced. Thus, primary antibodies are introduced to the phosphorylated proteins.

(13) Cleaning (not shown)

(14) Introduction of the second antibody (FIG. 7 (i))

Using a method similar to items (2), (5), (6), (8) and (9), the reagent liquid 125-2 as the fluorescent dye-conjugated secondary antibody liquid was supplied by using the pipette tip 151-2, allowed to stand for one hour, and the reagent liquid 125-2 was reduced. By this treatment, the fluorescent dye-conjugated secondary antibody is introduced to the protein to be phosphorylated, and thus the introduction of the labeling substance to the spot including the protein that reacts with the phosphorylase contained in the specimen is completed.

(15) Cleaning (not shown)

(16) Observe the supply of liquid (FIG. 8 (j))

The observation liquid 107 is supplied using a method similar to items (2), (5) and (6).

Next, a fluorescence detection step will be described with reference to fig. 9. Fig. 9 is a view for illustrating an optical detection step.

The observation liquid 107 is loaded inside the frame 105 of the array plate 100.

Light emitted from the semiconductor laser 201 passes through the collimator lens 203 and the band-pass filter 205, is reflected by the long-pass filter 207, and passes through the objective lens 209 to be collected to the spot 103 on the first surface 101-1 of the array plate 100.

The fluorescent substance is introduced into a spot including a protein that reacts with phosphorylase contained in the specimen, and thus the spot emits fluorescence. The fluorescence emitted from the spot 103 passes through the objective lens 209 and the long-pass filter 207, and is collected by the imaging lens 213. Light passing through the pinhole 215 is detected by a photomultiplier tube 217.

The optical unit 221 is scanned along the array plate 100 by the horizontal stage 223, so that a fluorescent two-dimensional image of the array plate 100 is acquired.

In this example, as the observation liquid, a glycerin solution having a refractive index of 1.4 or more, which is close to that of glass, was used. Therefore, stray light caused by reflected light is reduced, and the SN ratio can be improved, as compared with the case where the observation liquid is not used. Further, from the step of bringing the specimen and the plurality of spots into contact with each other to the step of acquiring optical information of the plurality of spots in a state where the spots are in contact with the observation liquid, these steps are continuously performed without going through the drying step, and thus an improvement in throughput can be expected. Further, adverse effects caused by drying unevenness can be suppressed.

The structure of the frame serving as the holding mechanism is described with reference to fig. 10.

Fig. 10 (a-1), 10 (b-1) and 10 (c-1) are top views of the array plate 100, and fig. 10 (a-2), 10 (b-2) and 10 (c-2) are cross-sectional views of dotted circle parts of the respective top views.

For the array plate 100 shown in fig. 10 (a-1) and 10 (a-2), as shown in fig. 10 (b-1) and 10 (b-2), a quadrangular rubber 501 and a frame 503 are installed. The frame 503 has a concave portion to be in contact with the rubber 501. Further, as shown in (c-1) of fig. 10 and (c-2) of fig. 10, the frame 503 and the base plate 101 are formed by fitting the supporting member 505 to the side wall portion such that the rubber is pressed. A recess may be formed in the frame 503 so that the supporting member 505 may be easily assembled.

The structure of the frame is not limited thereto, and the frame may have any shape as long as the frame can contain a liquid.

Example 2

A case is described in which the target of the specimen is an antigen and determination of the presence or absence of the antigen and/or quantification of the antigen is performed. In this case, it is effective to use array plates including spots each containing an antibody specifically recognizing an antigen as a biological substance. Hereinafter, such an array plate is also referred to as an "antibody array plate".

A specimen extracted from a living body, a reagent liquid containing a biotinylated detection antibody mixture and fluorescent labeled streptavidin, as a reagent liquid, a cleaning liquid, and an observation liquid are prepared.

The reaction was carried out as follows.

First, a specimen is brought into contact with a first surface of an antibody array plate. In this state, the antibody array plate is stationary for a predetermined period of time, so that the antibodies contained in the antibody array plate react with the specific antigen.

Next, after cleaning, a mixture of biotin and biotinylated detection antibodies that bind to antibodies specific for the antigen is disposed on the first surface of the antibody array plate.

Further, after the cleaning, fluorescence-labeled streptavidin as a fluorescent substance that binds to streptavidin having a property of binding to biotin is supplied to the array plate, and the fluorescent substance is introduced to spots that have reacted with the target.

Finally, the liquid on the first surface of the array plate was replaced with the observation liquid, and the optical information was obtained similarly to example 1.

In this example, detection and quantification of the presence or absence of a target contained in a specimen are effectively performed.

Similarly to example 1, in this example, as the observation liquid, a glycerin solution having a refractive index of 1.4 or more, which is close to that of glass, was used. Therefore, stray light caused by reflected light is reduced, and the SN ratio can be improved, as compared with the case where the observation liquid is not used. Further, from the step of bringing the specimen and the plurality of spots into contact with each other to the step of acquiring optical information of the plurality of spots in a state where the spots are in contact with the observation liquid, these steps are continuously performed without going through the drying step, and thus an improvement in throughput can be expected.

Further, adverse effects caused by drying unevenness can be suppressed.

Example 3

For the purpose of acquiring information on the immunity of a specimen, an array plate including spots containing a plurality of antigen molecules as biological substances is used. Hereinafter, this array plate is also referred to as an "antigen array plate".

When an antibody specific for the antigen of the spot is present in the specimen, the antibody reacts with the antigen.

In this example, the specimen is human serum and the target is an IgG antibody. A specimen, a reagent liquid containing a mixture of biotinylated anti-human IgG antibodies as a reagent liquid and a reagent liquid containing fluorescent labeled streptavidin, a cleaning liquid, and an observation liquid are prepared.

The treatment is as follows.

First, a specimen is brought into contact with a first surface of an antigen array plate. In this state, the antigen array plate is stationary for a predetermined period of time, so that the antigen of the antigen array plate reacts with the antibody contained in the specimen.

Next, after cleaning, a biotinylated anti-human IgG antibody mixture is disposed on the first surface of the antibody array plate such that secondary antibodies bind to the antigen-bound antibodies.

Further, after cleaning, a fluorescent labeled streptavidin formed of streptavidin having a strong binding property to biotin is supplied so that the second antibody is labeled.

Finally, the liquid on the antigen array plate was replaced with the observation liquid, and fluorescence detection was performed similarly to example 1.

By this example, the immune status of the specimen can be grasped effectively.

Further, adverse effects caused by drying unevenness can be suppressed.

Example 4

When analyzing the activity of protein kinase, a protein array plate is used. The protein array plate is an array plate including spots each containing a protein serving as a matrix of protein kinase as a biological substance. As another example, a peptide array plate is used. The peptide array plate is an array plate including spots each containing a peptide having a matrix site sequence as a biological substance immobilized on the spots.

The processing and effects in this case are the same as those described in example 1.

Example 5

With reference to fig. 11, an optical system in an analysis device according to the present invention is described.

Fig. 11 (a) shows the same arrangement as that described in example 1, in which fluorescence is detected from the back surface side of the slide glass as the second surface side of the array plate. The arrangement of the optical system is not limited thereto, and may take various forms as described below.

In fig. 11 (b), the objective lens 309 is arranged on the first surface 101-1 side of the array plate 100, that is, on the front surface side of the substrate 101, and fluorescence is detected from the same side. Even with this arrangement, stray light caused by reflected light can be reduced as compared with the case where no observation liquid is used, and thus the SN ratio can be improved.

Further, in (c) of fig. 11, the array plate 100 is arranged in a transparent container 401, and the container 401 is filled with the observation liquid 107. An objective lens 409 is provided, and fluorescence transmitted through the substrate 101 and the container 401 is detected. Even with this arrangement, stray light caused by reflected light can be reduced as compared with the case where no observation liquid is used, and thus the SN ratio can be improved. Furthermore, there is no need to provide a frame on the array plate 100, and thus there is an advantage in that spots are arranged on a slide glass without any region limitation, as compared with fig. 11 (a) and 11 (b). Further, as shown in (d) of FIG. 11, fluorescence may be detected from the first surface 101-1 side of the array plate.

Example 6

Fig. 12 and 13 are views for illustrating a method of reducing reagent liquid by using a substrate tilting mechanism in the specimen analysis apparatus according to the present invention.

Fig. 12 is a view for illustrating a support base for supporting an array plate. Fig. 12 (a) is a top view including a support portion 601, an array plate support portion 603, a plate spring 605, and a support portion 607. The support base or array plate support section 603 shown in fig. 12 (a) and the subsequent drawings is an example of the placement unit 1001. Fig. 12 (B) is a sectional view of the portion A-A, and fig. 12 (c) is a sectional view of the portion B-B.

Fig. 13 is a view for illustrating a method of discharging liquid.

In fig. 13 (a), the array plate supporting portion 603 supports the array plate 100 in a state of containing the reagent liquid 126. A vertical stage (not shown) is controlled so that the pipette tip 151 moves downward. Thus, the inside of the frame 105 is pressed by the distal end of the pipette tip 151. As a result, as shown in (b) of fig. 13, the array plate 100 is tilted.

Next, as shown in (c) of fig. 13, a pipette operation unit (not shown) is controlled so that the pipette tip 151 aspirates the reagent liquid 126.

Then, a vertical stage (not shown) is controlled so that the pipette tip 151 moves upward. At this time, the array plate 100 returns to its original position due to the spring force of the plate spring 605.

According to the present example, the amount of liquid remaining when the liquid is discharged decreases. As a result, effects such as process stabilization and reduction in the number of cleaning times can be obtained.

Example 7

Fig. 14 is a view for illustrating a second substrate tilting mechanism in the specimen analysis apparatus according to the present invention.

Fig. 14 is a view for illustrating a support base for supporting an array plate. Fig. 14 (a) is a top view including a support portion 601, an array plate support portion 701, a rotation shaft 703, a torsion spring 705, and a support portion 607. Fig. 14 (B) is a sectional view of the portion A-A, and fig. 14 (c) is a sectional view of the portion B-B.

The operation of the method of discharging the liquid was similar to that of example 8.

According to the present example, similarly to example 8, the amount of liquid remaining when the reagent liquid was discharged was reduced. As a result, effects such as process stabilization and reduction in the number of cleaning times can be obtained.

Example 8

Fig. 15 is a view for illustrating a third substrate tilting mechanism in the specimen analysis apparatus according to the present invention.

Fig. 15 is a view for illustrating a support base for supporting an array plate. The basic structure is similar to that of fig. 14. Fig. 15 (a) is a plan view. Fig. 15 (a) is different from fig. 14 in that openings formed in an array plate supporting portion 801 for supporting an array plate are rotated in a plane, that is, sides of the openings are not parallel to sides of the array plate supporting portion 801.

Fig. 15 (b) is a plan view of the case where the array board 100 is mounted.

The operation of the method of discharging the reagent liquid was similar to that of example 6.

According to the present example, similarly to example 6, the amount of liquid remaining when the reagent liquid was discharged was reduced. As a result, effects such as process stabilization and reduction in the number of cleaning times can be obtained.

In addition, when the array plate is tilted, the lowest point reaches the inside of the frame. Thus, the reagent liquid was sucked at the lowest point, and thus the amount of residual liquid was further reduced as compared with example 6.

Example 9

Fig. 16 is a view for showing the structure of a second frame of the specimen analysis apparatus according to the present invention.

The basic structure is similar to that of fig. 10.

Fig. 16 is different from fig. 10 in that pentagonal rubber 901 and pentagonal frame 903 are mounted to the array panel 100. The pentagonal frame 903 has a concave portion in contact with the rubber 901. In this way, even when the array plate is tilted by the method of example 8 or example 9, the lowest point enters the inside of the frame.

Example 10

By using the method according to the present application, phosphorylase contained in a specimen is detected. The phosphorylated substrate was immobilized on circular spots of 1mm diameter using a glass slide as a substrate. The circular spots are arranged at a pitch of 2 mm. The specimen containing the phosphorylase is contacted with the spot. The labeling substance is then introduced by identifying the phosphorylated spots with a dye (Alexa Fluor 680). The observation liquid is brought into contact with the first surface of the array plate including the spots, and fluorescence generated by applying a semiconductor laser having a wavelength of 670nm is measured in a state where the observation liquid is in contact with the first surface, so that an image is created. The results are shown in FIG. 17. Fig. 17 shows optical information 800 obtained in an example of the application. The optical information 800 includes a plurality of spots of optical information 803-11 to 803-18 and a background 802. Fig. 17 further illustrates a schematic diagram 900 of an array plate used in this example. Schematic 900 includes a plurality of spots 903-11 through 103-36 and a slide 901.

The present disclosure is not limited to the above-described embodiments, and various changes and modifications may be made without departing from the spirit and scope of the present disclosure. To disclose the scope of the present disclosure, the following claims are appended hereto.

The present application claims priority based on japanese patent application No. 2021-115910 filed on day 2021, 7 and 13 and japanese patent application No. 2022-094140 filed on day 2022, 6 and 10, the entire contents of which are incorporated herein by reference.

List of reference numerals

S1000: first identification step

S2000: a second identification step

S100: specimen introduction step

S200: sample amount reducing step

S300: observing the liquid introduction step

S400: acquisition step

100: Array plate

101: Substrate board

103: Speckle pattern

105: Frame

107: Observing the liquid

143: Specimen

1001: Placement unit

1002: Liquid feeder/discharger

1003: Optical system

1004: Control system

1013: Mobile unit

1014: Scanner

1015: Retainer

2000: Target(s)

2001: Marking substance

2002: Biological material

3001: Specimen introducing mechanism

3002: Specimen quantity reducing mechanism

3003: Observing liquid introducing mechanism

3004: Acquisition mechanism

3005: First identification mechanism

3006: Second identification mechanism

4001: Analysis device

4003: Door

4005: Emergency stop switch

4051: Liquid crystal display device

4053: Keyboard with keyboard body

4055: Mouse with mouse body

4101: Work table

Claims

1. An analysis method for analyzing a specimen containing a target by using an array plate including a plurality of spots each containing a biological substance on one surface thereof, the analysis method comprising:

A specimen introducing step of bringing a specimen and the plurality of spots into contact with each other to react a portion of the plurality of spots and a target with each other;

A specimen amount reducing step of reducing an amount of a specimen in contact with the plurality of spots;

An observation liquid introducing step of bringing the one surface into contact with an observation liquid so that the plurality of spots are brought 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 an observation liquid,

The analysis method further includes at least any one of a first identification step of identifying the target and a second identification step of identifying the portion of the plurality of spots that react with the target prior to the specimen introduction step, the second identification step being performed after the specimen amount reduction step.

2. The method of analysis according to claim 1,

Wherein the analysis method comprises a first identification step, and

Wherein the first labeling step includes performing labeling by using a fluorescent substance.

3. The method of analysis according to claim 1,

Wherein the analysis method comprises a second identification step, and

Wherein the second labeling step includes performing labeling by using a fluorescent substance.

4. The analysis method according to any one of claims 1 to 3, wherein the obtaining step includes:

Applying excitation light to the plurality of spots; and

At least any one of light intensity information and spectrum information of fluorescence emitted from the plurality of spots is acquired as the optical information.

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

6. The analysis method according to claim 4 or 5, wherein the array plate has a transmissivity to a wavelength of the excitation light.

7. The assay of claim 4 or 6, wherein the array plate is transmissive to the wavelength of the fluorescence.

8. The analysis method according to any one of claims 4 to 7, wherein the acquiring step includes:

applying the excitation light to the plurality of spots from a back surface side of the one surface; and

Fluorescence is detected from the back surface side.

9. The analysis method according to any one of claims 1 to 8, wherein the observation liquid has a refractive index of 1.40 or more and 1.46 or less.

10. The analysis method according to any one of claims 1 to 9, wherein the observation liquid contains 40vol% or more and 90vol% or less of glycerin.

11. The assay of claim 1 and any one of claims 3 to 10, wherein the second identifying step comprises contacting at least any one reagent liquid with the plurality of spots with each other.

12. The assay of any one of claims 1 to 11, wherein the biological substance comprises at least any one of a nucleic acid, a peptide and a protein.

13. The assay of any one of claims 1 to 11, wherein the biological substance comprises a phosphorylase matrix.

14. The assay of claim 13, wherein the reagent liquid comprises a phosphorylation site recognition material.

15. The analysis method according to any one of claims 1 to 14, further comprising a specimen information acquisition step of acquiring information about a specimen based on the optical information.

16. The analysis method according to any one of claims 1 to 15, wherein the observation liquid introduction step is further used as a part of the specimen amount reduction step.

17. The method of any one of claims 1 to 16, wherein a specimen in contact with the plurality of spots is replaced with an observation liquid in contact with the plurality of spots.

18. An analysis device for analyzing a specimen containing a target by using an array plate including a plurality of spots each containing a biological substance on one surface thereof, the analysis device comprising:

A specimen introducing mechanism configured to bring a specimen and the plurality of spots into contact with each other;

A specimen amount reduction mechanism configured to reduce an amount of specimen in contact with the plurality of spots;

An observation liquid introducing mechanism configured to bring the one surface into contact with an observation liquid so that the plurality of spots are brought into contact with the observation liquid; and

An acquisition mechanism configured to acquire optical information of the plurality of spots in a state where the plurality of spots are in contact with an observation liquid,

The analysis device further includes at least any one of a first identification mechanism configured to operate and identify the target prior to operation of the specimen introduction mechanism and a second identification mechanism configured to operate and identify a portion of the plurality of spots that react with the target after operation of the specimen amount reduction mechanism.

19. The apparatus according to claim 18,

Wherein the array plate has a transmissivity to excitation light to be used in the acquisition mechanism, and

Wherein the acquisition mechanism is configured to:

Applying the excitation light to the plurality of spots from a back surface side of the one surface of the array plate; and

The optical information of fluorescence emitted from the plurality of spots is acquired from the back surface side.

20. The analysis device of claim 18 or 19, further comprising a holding mechanism configured to hold the observation liquid such that the plurality of spots are in contact with the observation liquid.

21. The analysis device of claim 20, further comprising a tilting mechanism configured to tilt at least any one of the array plate and the holding mechanism relative to a horizontal plane.

22. The analysis device of claim 21, wherein the tilting mechanism is configured to press one of the array plate and the holding mechanism vertically downward.

23. The analysis device of claim 21 or 22, wherein the tilting mechanism comprises at least any one of a pipette tip and a needle.

24. The analysis device of any one of claims 18 to 23, further comprising a mechanism configured to obtain information about a specimen based on the optical information.

25. An analysis device, comprising:

a placement unit on which an array plate including a plurality of spots each containing a biological substance on one surface thereof is to be placed;

a holder configured to hold a liquid such that the one surface and the liquid are in contact with each other;

a liquid feeder/discharger configured to introduce the liquid to a liquid-holding region of the holder or discharge the liquid held by the holder;

a moving unit configured to move the liquid feeder/ejector relative to the placement unit;

An optical system configured to apply light to the plurality of spots to detect light emitted from the plurality of spots;

a scanner configured to move the optical system relative to the placement unit; and

A controller configured to control a time at which each of the following steps is performed: a specimen introducing step of bringing a specimen and the plurality of spots into contact with each other to cause a portion of the plurality of spots and a target contained in the specimen to react with each other, a specimen amount reducing step of reducing an amount of the specimen in contact with the plurality of spots, an observation liquid introducing step of bringing the one surface into contact with an observation liquid so that the plurality of spots are in contact with the observation liquid, and an acquiring step of acquiring optical information of the plurality of spots in a state in which the plurality of spots are in contact with the observation liquid, the controller being further configured to control a time to perform at least any one of a first identifying step of identifying a target and a second identifying step of identifying the portion of the plurality of spots reacting with the target, the first identifying step being performed before the specimen introducing step, the second identifying step being performed after the specimen amount reducing step.

26. The analysis device of claim 25, wherein the controller is configured to control the placement unit, the liquid feeder/ejector, the movement unit, the optical system, and the scanner to perform the specimen introducing step, the specimen amount reducing step, the observation liquid introducing step, and the first identifying step.

27. The analysis device according to claim 26, wherein the controller is configured to perform at least any one of the specimen introducing step, the specimen amount reducing step, the observation liquid introducing step, and the first identifying step for a second array plate different from the first array plate in parallel with the execution of the acquiring step for the first array plate.

28. The analysis device of claim 25, wherein the controller is configured to control the placement unit, the liquid feeder/ejector, the movement unit, the optical system, and the scanner to perform the specimen introducing step, the specimen amount reducing step, the observation liquid introducing step, and the second identifying step.

29. The analysis device according to claim 28, wherein the controller is configured to perform at least any one of the specimen introducing step, the specimen amount reducing step, the observation liquid introducing step, and the second identifying step for a second array plate different from the first array plate in parallel with the execution of the acquiring step for the first array plate.

30. A program for causing a computer to execute the steps of the analysis method according to any one of claims 1 to 17.