Preparation method of biological sample for assay, preparation apparatus of biological sample for assay, target substance identification method, and target substance identification apparatus

Abstract

To provide a method of preparing a biological sample for an assay that can reduce an application amount of a reaction solution for identifying a target substance in a biological sample.SOLUTION: A method of preparing a biological sample for an assay includes determining a contour of the biological sample on a substrate, using an ink jet liquid ejection head to form a holding portion for holding at least part of the reaction solution including a chemical compound that can be specifically coupled to a target substance in the biological sample inside the contour on the substrate along the contour, and applying the reaction solution to the biological sample.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for preparing a biological sample for analysis, an apparatus for preparing a biological sample for analysis, a method for identifying a target substance in a biological sample, and an apparatus for identifying a target substance.

Background Art

[0002] In pathology, tests related to diseases are performed to find the cause of the disease. Detection of target substances by staining with dyes such as HE staining, staining in immunostaining or in situ hybridization, etc. is often required in pathology as an important test for determining a treatment policy. In a pathological examination, cells or tissue pieces taken from a living body are attached to a substrate such as a slide glass to form a sample. Thereafter, the sample is brought into contact with a predetermined detection solution to stain the cells or tissue pieces, and the target substance is detected.

[0003] The detection solution used in pathological examinations is expensive. In particular, antibodies used in immunostaining and probes used in in situ hybridization are very expensive, so detection solutions containing these are often used in small amounts. For this reason, there is a desire to accurately place the detection solution on a biological sample containing the target substance to be stained and to maintain that state as much as possible.

[0004] As a solution to such problems, a pen (pap pen) for forming a water-repellent frame on a slide glass is known. For a biological sample placed on a slide glass, a water-repellent frame is formed using a pap pen so as to surround the biological sample, and the detection solution is applied within the frame. Thereby, by suppressing the movement of the detection solution to a portion where there is no target to be stained, staining with a small amount of the detection solution can be efficiently performed.

[0005] In addition, Patent Document 1 discloses a tissue staining sheet having through-holes that form independent compartments. By adhering this tissue staining sheet to a slide glass, it is disclosed that a plurality of compartments are formed on the slide glass, suppressing the outflow of antibodies and probes to other compartments.

Prior Art Document

Patent Document

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] Since a felt-tip pen forms a water-repellent frame by handwriting, it is difficult to create a fine frame, so it has been difficult to further reduce the amount of detection liquid (hereinafter also referred to as "reaction liquid") applied.

[0008] Also, in the tissue staining sheet described in Patent Document 1, the shape and size of the through-holes for forming compartments in advance are determined. Therefore, it has been impossible to change the shape and size of the through-holes according to the biological sample, and it has been difficult to further reduce the amount of the reaction liquid applied.

[0009] Therefore, an object of the present invention is to provide a method for preparing a biological sample for analysis, a device for preparing a biological sample for analysis, a method for identifying a target substance, and a device for identifying a target substance, which can reduce the amount of reaction liquid applied for identifying a target substance in a biological sample.

Means for Solving the Problems

[0010] According to the present invention, there is provided a method for preparing a biological sample for analysis, comprising: a step of specifying the contour of the biological sample on a substrate; A step of forming, on the substrate along the contour, a holding portion for holding at least a part of a reaction solution containing a compound capable of specifically binding to a target substance in the biological sample inside the contour using an inkjet liquid ejection head; A step of applying the reaction solution to the biological sample to obtain the biological sample for analysis; There is provided a method for preparing a biological sample for analysis, characterized by comprising the above steps.

[0011] According to the present invention, there is provided an apparatus for preparing a biological sample for analysis, comprising: Contour specifying means for specifying the contour of the biological sample on the substrate; Liquid ejection means having an inkjet liquid ejection head for forming, on the substrate along the contour, a holding portion for holding at least a part of a reaction solution containing a compound capable of specifically binding to a target substance in the biological sample inside the contour; Reaction solution applying means for applying the reaction solution to the biological sample to obtain the biological sample for analysis; There is provided an apparatus for preparing a biological sample for analysis, characterized by comprising the above components.

[0012] According to the present invention, there is provided a method for identifying a target substance in a biological sample, comprising: A step of specifying the contour of the biological sample on the substrate; A step of forming, on the substrate along the contour, a holding portion for holding at least a part of a reaction solution containing a compound capable of specifically binding to a target substance in the biological sample inside the contour using an inkjet liquid ejection head; A step of applying the reaction solution to the biological sample; A step of identifying the target substance by detecting the compound bound to the target substance; There is provided a method for identifying a target substance, characterized by comprising the above steps.

[0013] Further, according to the present invention, there is provided an apparatus for identifying a target substance in a biological sample, comprising: Contour specifying means for specifying the contour of the biological sample on the substrate, Liquid discharge means having an inkjet liquid discharge head for forming a holding part on the substrate for holding at least a part of a reaction liquid containing a compound capable of specifically binding to a target substance in the biological sample inside the contour along the contour, Reaction liquid applying means for applying the reaction liquid to the biological sample, Discriminating means for detecting the compound bound to the target substance, There is provided a target substance discrimination device characterized by comprising the above.

Advantages of the Invention

[0014] According to the present invention, it is possible to provide a method for preparing a biological sample for analysis, a device for preparing a biological sample for analysis, a method for discriminating a target substance, and a discrimination device capable of reducing the amount of a reaction liquid applied for discriminating a target substance in a biological sample.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0016] Hereinafter, the present invention will be described in detail with reference to preferred embodiments. In the present invention, when the compound is a salt, although the salt exists dissociated into ions in the reaction liquid or the liquid composition, for convenience, it is expressed as "containing a salt". Further, physical property values are values at normal temperature (25°C) unless otherwise specified.

[0017] In order to reduce the amount of reaction solution applied for identifying a target substance in a biological sample, the present inventors studied the formation of a holding portion for holding the reaction solution on a substrate.

[0018] As a result, the present inventors found that it is important to form a holding portion along the contour of the biological sample on the substrate after specifying the contour of the biological sample on the substrate, and thus arrived at the present invention.

[0019] In the present invention, first, the contour of the biological sample on the substrate is specified. Then, based on the information of the specified contour, a holding portion is formed along the contour of the biological sample on the substrate using an inkjet liquid ejection head. Since the contour of the biological sample is specified in advance, even if the contours of the biological samples are different from each other, a holding portion adapted to the contour can be formed for each biological sample. Further, since the holding portion is formed using an inkjet liquid ejection head, it is possible to form a fine holding portion. Thereby, the movement of the reaction solution to the area where there is no biological sample on the substrate due to the wet spread of the applied reaction solution is suppressed, and according to the contour of the biological sample, the amount of the reaction solution not involved in the identification of the target substance can be reduced as much as possible. Therefore, even if the amount of the reaction solution applied is reduced to the minimum amount necessary for identifying the target substance, the target substance in the biological sample can be sufficiently identified.

[0020] <<Method for preparing a biological sample for analysis, apparatus for preparing a biological sample for analysis, method for identifying a target substance, and apparatus for identifying a target substance>> The method for preparing a biological sample for analysis of the present invention (hereinafter simply referred to as "preparation method") has the following three steps. (a1) A step of specifying the contour of the biological sample on the substrate. (b1) A step of forming, on the substrate, a holding portion for holding at least a part of a reaction solution containing a compound capable of specifically binding to a target substance in the biological sample inside the contour along the contour using an inkjet liquid ejection head. (c1) A step of applying the reaction solution to the biological sample to obtain the biological sample for analysis.

[0021] In addition, the preparation apparatus for a biological sample for analysis according to the present invention (hereinafter, also simply referred to as the "preparation apparatus") has the following three means. (A1) A contour specifying means for specifying the contour of the biological sample on a substrate. (B1) A liquid discharge means having an inkjet-type liquid discharge head for forming a holding portion on the substrate for holding at least a part of a reaction solution containing a compound capable of specifically binding to a target substance in the biological sample inside the contour along the contour. (C1) A reaction solution applying means for applying the reaction solution to the biological sample in order to obtain the biological sample for analysis.

[0022] The method for identifying a target substance according to the present invention (hereinafter, also simply referred to as the "identification method") has the following four steps. (a2) A step of specifying the contour of the biological sample on a substrate. (b2) A step of forming, on the substrate along the contour, a holding portion for holding at least a part of a reaction solution containing a compound capable of specifically binding to a target substance in the biological sample inside the contour by using an inkjet-type liquid discharge head. (c2) A step of applying the reaction solution to the biological sample. (d2) A step of identifying the target substance by detecting the compound bound to the target substance.

[0023] In addition, the identification apparatus for a target substance according to the present invention (hereinafter, also simply referred to as the "identification apparatus") has the following four means. (A2) A contour specifying means for specifying the contour of the biological sample on a substrate. (B2) A liquid discharge means having an inkjet-type liquid discharge head for forming a holding portion on the substrate for holding at least a part of a reaction solution containing a compound capable of specifically binding to a target substance in the biological sample inside the contour along the contour. (C2) A reaction solution applying means for applying the reaction solution to the biological sample. (D2) An identification means for detecting the compound bound to the target substance.

[0024] The method for preparing a biological sample for analysis has substantially the same configuration as the method for identifying a target substance, except that it does not have a step of identifying the target substance by detecting the compound bound to the target substance. Further, the apparatus for preparing a biological sample for analysis has substantially the same configuration as the apparatus for identifying a target substance, except that it does not have an identification means for detecting the compound bound to the target substance.

[0025] <Apparatus for Preparing a Biological Sample for Analysis> Hereinafter, the apparatus for preparing a biological sample for analysis will be described with reference to the drawings. FIG. 1 is a schematic diagram showing an embodiment of the preparation apparatus of the present invention, and FIG. 2 is a schematic diagram showing each step in an embodiment of the preparation method used in an embodiment of the preparation apparatus. The preparation apparatus 100 of the present embodiment includes a contour identification device 101 as a contour identification means, a liquid discharge device 102 as a liquid discharge means, and a reaction solution application device 103 as a reaction solution application means. The substrate 105 on which the biological sample 104 is placed is conveyed in the direction of arrow A by a conveyance device (not shown). Note that the conveyance device may be provided in the preparation apparatus or may exist as a device separate from the preparation apparatus.

[0026] The contour specifying device 101 is present above the substrate 105 and specifies the contour of the biological sample 104 on the substrate 105 (Fig. 2(a)). The liquid ejection device 102 has an inkjet-type liquid ejection head. Then, based on the information regarding the contour of the biological sample 104 specified by the contour specifying device 101, a holding portion 106 for holding at least a part of the reaction liquid inside the contour is formed along this contour on the substrate 105 (Fig. 2(b)). After the holding portion 106 is formed by this liquid ejection device 102, the reaction liquid 107 is applied to the biological sample surrounded by the holding portion 106 by the reaction liquid applying device 103 to bind the compound contained in the reaction liquid to the target substance (Fig. 2(c)). As a result, the target substance in the biological sample can be made detectable, and thus the target substance can be identified. That is, a biological sample for analysis can be obtained. In Fig. 1, an example in which the substrate 105 is conveyed by a conveying device is shown, but the substrate 105 may be fixed. When the substrate 105 is fixed, each of the contour specifying device 101, the liquid ejection device 102, and the reaction liquid applying device 103 is further equipped with a conveying device, and the contour specifying device 101, the liquid ejection device 102, and the reaction liquid applying device 103 are in a movable state. Then, by moving the contour specifying device 101, the liquid ejection device 102, and the reaction liquid applying device 103, the specification of the contour of the biological sample 104, the formation of the holding portion 106, and the application of the reaction liquid 107 are performed.

[0027] Hereinafter, each configuration of the preparation device for a biological sample for analysis will be described.

[0028] [Contour Specifying Device] Since the biological sample 104 on the substrate 105 is often not in a regular shape such as a rectangle or a circle, it is necessary to accurately identify the contour. Therefore, the contour identification device 101 identifies the contour of the biological sample 104. The contour identification device 101 is not particularly limited as long as it can identify the contour of the biological sample 104. Among them, it is preferable that the contour identification device 101 optically identifies the contour of the biological sample 104. Specifically, it is preferable to optically distinguish the biological sample 104 from the air phase around it. Examples of the contour identification device that optically identifies the contour include known devices such as a photographing device having a lens and a sensor that utilizes optical reflection. The contour identification device 101 is preferably provided in the preparation device so as to be able to scan in order to identify the contours of biological samples in a wide range.

[0029] In addition, the data regarding the contour of the biological sample 104 obtained by the contour identification device 101 is sent to the liquid ejection device 102. There is no particular limitation on the method of sending the data regarding this contour to the liquid ejection device 102, and known methods can be used. For example, by electrical means, the data regarding the contour is sent to a computer, and in the computer, this data is converted into data regarding the ejection patterning when forming a holding portion by the liquid ejection device 102, and the data can be sent to the liquid ejection device 102.

[0030] 〔Biological Sample〕 Examples of biological samples include cultured cells, body fluids of animals (e.g., blood, serum, plasma, lymph, sweat, saliva, urine, etc.), hair, excrement, organs, tissues, or animals and plants themselves, or samples obtained by fixing them and embedding them in paraffin, dried bodies, and the like.

[0031] 〔Substrate〕 In pathological staining, cell staining is often performed after placing a thinly sliced biological sample on a slide glass as the substrate, but in the present invention, the substrate is not limited to a slide glass. Specifically, examples of the substrate include slide glass, paper, plastic, metal, rubber, and ceramic.

[0032] [Liquid ejection device] The liquid ejection device 102 has an inkjet-type liquid ejection head. Then, based on the data of the contour of the biological sample 104 obtained by the contour identification device 101, a holding part 106 for holding the reaction liquid is formed. By using an inkjet-type liquid ejection head, a liquid composition for forming the holding part in the picoliter level amount (hereinafter, also simply referred to as the liquid composition) can be applied, so that a fine holding part 106 can be formed along the contour of the biological sample. Note that the holding part 106 only needs to be formed so that at least a part of the reaction liquid can be held inside the contour. Therefore, the liquid composition does not necessarily have to be solidified on the base material after being applied to the base material. For example, even if the holding part formed by the liquid composition is not solidified, as long as the reaction liquid can be held inside the contour of the biological sample and the position of the holding part does not move so that the target substance can be accurately identified by the thickening of the liquid composition or the like. Examples of the inkjet-type liquid ejection head include a form in which liquid is ejected by causing film boiling in the liquid by an electro-thermal converter to form bubbles, a form in which liquid is ejected by an electro-mechanical converter, and a form in which liquid is ejected using static electricity. In the present invention, a known inkjet-type liquid ejection head can be used. Among them, from the viewpoint of ejecting liquid at high speed and high density in particular, a liquid ejection head using an electro-thermal converter is preferable. In the inkjet-type liquid ejection head, an image signal corresponding to the shape of the holding part can be received, and the application amount of the liquid composition required for each position can be applied. Further, the liquid ejection device may include, in addition to the liquid ejection head, a cap or the like for suppressing the evaporation of the liquid composition from the ejection port of the liquid ejection head.

[0033] The holding portion 106 only needs to be formed along the contour of the biological sample 104, and it is not necessarily required to be formed such that the contour of the biological sample 104 and the holding portion 106 are in contact. However, from the perspective of reducing the amount of reaction solution applied, it is preferable that the holding portion 106 is formed along the contour of the biological sample 104 so as to be in contact therewith. Further, the holding portion 106 may be formed by discontinuous dots, but from the perspective of reducing the amount of reaction solution applied, it is preferable that the holding portion 106 is continuously formed so as to cover (surround) the periphery of the contour of the biological sample 104. In the present specification, a holding portion formed so as to continuously cover the periphery of the contour of a biological sample is also referred to as a partition wall.

[0034] The holding portion 106 is preferably a partition wall that partitions a space for holding the reaction solution inside the contour of the biological sample. FIG. 3 is a cross-sectional view of a biological sample on a substrate when a partition wall is formed as the holding portion. As shown in FIG. 3, since the holding portion 106 is a partition wall having a predetermined height, more reaction solution can be held inside the contour of the biological sample, and the reaction solution can more easily come into contact with the target substance in the biological sample, which is preferable. There is no particular limitation on the height of the partition wall, but as shown in FIG. 3, it is preferable that the minimum height of the partition wall from the surface of the substrate is higher than the maximum height from the surface of the substrate 105 of the biological sample 104, because the reaction solution can be more easily held inside the contour of the biological sample.

[0035] 〔Liquid Composition〕 The holding portion 106 is formed by the liquid composition applied by the liquid ejection device 102.

[0036] (Thickening Component) The liquid composition applied by the liquid discharge device 102 to form the holding portion 106 preferably contains a thickening component that thickens by reaction after being applied to the substrate. Thereby, the liquid composition applied to the substrate thickens, and as shown in FIGS. 2 and 3, a holding portion can be formed that accurately follows the contour of the biological sample on the substrate. Also, a partition wall that continuously covers the periphery of the contour of the biological sample and has a certain height can be formed. As a result, even when the surface tension of the reaction solution is low or the amount of the reaction solution is increased, it becomes possible to hold the reaction solution in the partition wall. Also, a crosslinked structure may be formed by this reaction. When a crosslinked structure is formed, it becomes easier to suppress the dissolution by the reaction solution applied in the next step and the influence of the reactivity of the reaction solution on the target substance. By this thickening component, not only is the holding portion thickened, but it may also be solidified.

[0037] The reaction of the liquid composition, after being applied to the substrate, includes methods of applying energy such as light and heat, methods of applying different liquid compositions, and the like. Among them, the holding portion 106 is preferably formed by using a plurality of liquid discharge heads and discharging liquid compositions containing constituent materials of different types of holding portions from the plurality of liquid discharge heads toward the substrate, and the different liquid compositions come into contact with each other on the substrate. In particular, as the constituent material of the holding portion contained in the liquid composition, it is preferable to contain a thickening component that can thicken by the contact of two different liquid compositions.

[0038] The thickening component preferably contains at least one component that interacts through at least one interaction selected from the group consisting of electrostatic interaction, hydrophobic interaction, and hydrogen bond formation. Among them, in particular, the thickening component is preferably an ionic component capable of causing electrostatic interaction. When an ionic component is used as the thickening component, it is preferable to separately contain different ionic components (cationic component and anionic component) in two liquid compositions (the first liquid composition and the second liquid composition). Thereby, the storage stability of the liquid composition and the applicability of the liquid composition can be improved. The ionic component preferably contains at least one selected from the group consisting of polyvalent metal salts, organic acids, and ionic resins, and more preferably contains an ionic resin. Examples of the ionic resin include cationic resins and anionic resins. Further, the liquid composition is preferably a first liquid composition containing a polyvalent metal salt, an organic acid, a cationic resin, or anionic resin, and a second liquid composition containing a cationic resin or anionic resin. More preferably, the liquid composition is a first liquid composition containing a cationic resin and a second liquid composition containing anionic resin. By the liquid composition containing the thickening component, the viscosity of the mixture obtained by bringing the two liquid compositions into contact can be increased, and the spreading of the mixture on the substrate can be suppressed. Thereby, a fine holding portion can be formed, it becomes possible to apply a variety of reaction solutions to one sample, and by suppressing the outflow of the reaction solution to the portion without an object to be identified, the reaction solution can be effectively used.

[0039] The liquid composition containing an organic acid has a buffering capacity in the acidic region (pH less than 7.0, preferably pH 2.0 to 5.0), and efficiently converts the anionic component present in the other liquid composition into the acid form and aggregates it. Examples of the organic acid include monocarboxylic acids such as formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, glycolic acid, lactic acid, salicylic acid, pyrrolecarboxylic acid, furancarboxylic acid, picolinic acid, nicotinic acid, thiophenecarboxylic acid, levulinic acid, coumaric acid, and their salts; dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, itaconic acid, sebacic acid, phthalic acid, malic acid, tartaric acid, and their salts and hydrogen salts; tricarboxylic acids such as citric acid, trimellitic acid, and their salts and hydrogen salts; tetracarboxylic acids such as pyromellitic acid and their salts and hydrogen salts; and the like. The content (mass %) of the organic acid in the liquid composition is preferably 1.0 mass % or more and 5.0 mass % or less based on the total mass of the liquid composition.

[0040] The polyvalent metal salt is a compound formed from metal ions with a valence of 2 or more (polyvalent metal ions) and anions. The polyvalent metal salt dissociates in the liquid composition to become polyvalent metal ions and aggregates the anionic component in the other liquid composition. The polyvalent metal salt may be a hydrate. Examples of the polyvalent metal ions constituting the polyvalent metal salt include divalent metal ions such as Ca 2+ 、Cu 2+ 、Ni 2+ 、Mg 2+ 、Sr 2+ 、Ba 2+ 、and Zn 2+ ; and trivalent metal ions such as Fe 3+ 、Cr 3+ 、Y 3+ 、and Al 3+ . Examples of the anions include Cl - 、Br - 、I - 、ClO - 、ClO2 - 、ClO3 - 、ClO4 - 、NO2 - 、NO3- 、SO4 2- 、CO3 2- 、HCO3 - 、PO4 3- 、HPO4 2- 、and H2PO4 - and other inorganic anions such as HCOO - 、(COO - )2、COOH(COO - )、CH3COO - 、C2H5COO - 、CH3CH(OH)COO - 、C2H4(COO - )2、C6H5COO - 、C6H4(COO - )2、and CH3SO3 - and other organic anions; can be mentioned. The content (mass%) in terms of polyvalent metal salt in the liquid composition is preferably 1.0 mass% or more and 20.0 mass% or less based on the total mass of the liquid composition.

[0041] Among the ionic resins, examples of the cationic resin include resins having a structure of primary to tertiary amines, resins having a structure of quaternary ammonium salts, etc. Specifically, resins having structures such as vinylamine, allylamine, vinylimidazole, vinylpyridine, dimethylaminoethyl methacrylate, ethyleneimine, guanidine, diallyldimethylammonium chloride, alkylamine·epichlorohydrin condensates can be mentioned. In order to enhance the solubility in the liquid composition, a cationic resin and an acidic compound can be used in combination, or a quaternization treatment of the cationic resin can be performed. When adding a cationic resin, the content (mass%) of the cationic resin in the liquid composition is preferably 0.1 mass% or more and 10.0 mass% or less based on the total mass of the liquid composition.

[0042] Among the ionic resins, examples of the anionic resin include resins having anionic groups such as carboxylic acid groups, sulfonic acid groups, and phosphonic acid groups. Specifically, acrylic resins and urethane resins having these anionic groups can be mentioned. When adding an anionic resin, the content (mass%) of the anionic resin in the liquid composition is preferably 0.1 mass% or more and 10.0 mass% or less based on the total mass of the liquid composition.

[0043] Both the first liquid composition and the second liquid composition may be applied before the reaction liquid is applied to the substrate. Also, there is no particular limitation on the order of application of the first liquid composition and the second liquid composition to the substrate.

[0044] When the thickening component is a polyvalent metal salt or an organic acid, it is preferable to increase the concentration of the thickening component in the second liquid composition relative to the hydrophobic component in the first liquid composition, or to increase the application amount of the second liquid composition. Thereby, it is easy to increase the viscosity of the mixed liquid of the first liquid composition and the second liquid composition, and the holding part can be efficiently formed. At this time, the application amount of the thickening component applied by the second liquid composition is within a range that does not affect the biological sample or the reaction liquid.

[0045] When the thickening component is a cationic resin or an anionic resin, it is preferable to apply the first liquid composition and the second liquid composition so that the thickening component in the first liquid composition and the thickening component in the second liquid composition are in an appropriate ratio. Thereby, the viscosity of the mixed liquid increases quickly, and the holding part can be efficiently formed.

[0046] When the thickening components contained in the first liquid composition and the second liquid composition are a cationic resin and an anionic resin, respectively, an appropriate ratio can be obtained by calculation from the amine value of the cationic resin and the acid value of the anionic resin.

[0047] Also, by changing the ratio of the application amount of the first liquid composition to the application amount of the second liquid composition and then measuring the viscosity of the mixed liquid, the ratio that can most effectively increase the viscosity can also be experimentally determined.

[0048] (Hydrophobic component) The liquid composition preferably has a surface tension different from that of the reaction liquid. When the reaction liquid is aqueous, the liquid composition preferably contains a hydrophobic component as a constituent material of the holding part. When the liquid composition is aqueous, it is preferable to impart hydrophilicity to the hydrophobic component contained in the liquid composition and dissolve or disperse it in the aqueous liquid composition for use.

[0049] The hydrophobic component contained in the liquid composition is not particularly limited as long as it has water repellency when forming the holding part. Specifically, the hydrophobic component is preferably at least one selected from the group consisting of alkyl ketene dimer, alkenyl succinic anhydride, rosin, wax, and ionic resins such as cationic resin and anionic resin. Also, as described above, the cationic resin and the anionic resin may also function as thickening components.

[0050] (Aqueous medium) The liquid composition preferably contains an aqueous medium. As the aqueous medium, the same one as the aqueous medium contained in the reaction liquid described later can be used.

[0051] (Water-soluble resin) The liquid composition preferably contains a water-soluble resin. A water-soluble resin refers to a resin that can be dissolved in water. The solubility of the water-soluble resin in water at 25°C is preferably 1% by mass or more. Also, by adding a water-soluble organic solvent to water, the solubility of the water-soluble resin can be increased and it can be dissolved in an aqueous medium for use. When the liquid composition further contains a water-soluble resin, the constituent material of the holding part and the water-soluble resin are mixed in the liquid composition. In that state, by bringing the first liquid composition and the second liquid composition into contact on a substrate, the constituent material of the holding part and the water-soluble resin combine and aggregate, rapidly increasing the viscosity, and a fine holding part can be formed. Therefore, the water-soluble resin contained in the liquid composition preferably has a property that contributes to an increase in viscosity when two different liquid compositions are mixed.

[0052] On the other hand, if the content of the water-soluble resin in the liquid composition is excessive, the water repellency of the holding part may decrease. Therefore, there is an appropriate range for the content of the water-soluble resin in the liquid composition. Regarding the content of the water-soluble resin in the liquid composition, by preliminarily examining the relative content ratio in the liquid composition with respect to the constituent material of the holding part to be contained together, an appropriate range that can achieve both an improvement in viscosity increase efficiency and suppression of a decrease in water repellency can be determined.

[0053] The water-soluble resin is preferably at least one selected from the group consisting of an anionic water-soluble resin and a cationic water-soluble resin.

[0054] (Other components) In addition to the above components, the liquid composition may contain various additives such as an antifoaming agent, a surfactant, a pH adjuster, a viscosity modifier, a rust inhibitor, a preservative, a fungicide, an antioxidant, and an anti-reducing agent, as required. Also, in order to make the holding part visible, color materials such as pigments and dyes may be used as additives as required.

[0055] [Reaction liquid applying device] As the reaction solution applying device, there is no particular limitation as long as it can apply a reaction solution containing a compound capable of specifically binding to a target substance in a biological sample, and known devices can be used. Among them, the reaction solution applying device preferably has an inkjet liquid ejection head that can apply the reaction solution by ejecting the reaction solution toward the biological sample. By ejecting the reaction solution from the inkjet liquid ejection head toward the biological sample, the reaction solution can be precisely applied to the biological sample.

[0056] 〔Reaction solution〕 The reaction solution contains a compound capable of specifically binding to a target substance in a biological sample. By this compound binding to the target substance, the target substance can be made in a distinguishable state.

[0057] (Staining agent) Examples of the compound capable of specifically binding to the target substance include a staining agent for staining the target substance. As the staining agent, a staining agent commonly used for biological samples can be applied.

[0058] For example, staining agents used in dye staining include hematoxylin, eosin, carmine, Coomassie blue, crystal violet, 2-(4 - amidinophenyl)-1H - indole - 6 - carboxamidine (DAPI), ethidium bromide, acid fuchsin, malachite green, methyl green, methylene blue, Nile blue, Nile red, rhodamine, and safranin, etc. In addition, staining agents used in various stainings such as PAS staining, Gram staining, and Giemsa staining can also be used.

[0059] In addition, examples of the staining agents used in immunostaining include, among antibodies, anti-immunoglobulin, anti-IgG, anti-IgM, anti-IgA, anti-IgE and other antibodies. Antibodies conjugated with enzymes such as peroxidase and alkaline phosphatase, and antibodies labeled with biotin, magnetic particles, fluorescent molecules, etc. can also be used. Antibody fragments such as Fab fragments digested with papain and F(ab’)2 fragments digested with pepsin can also be used. As the staining substrate, when the labeled antibody is peroxidase for example, diaminobenzidine and the like are used. Proteases, blocking agents, etc. used for pretreatment can also be used.

[0060] Examples of the staining agents used in in situ hybridization include DNA probes and RNA probes used as probes. Probes labeled with fluorescent molecules, digoxigenin, dinitrophenyl, etc. can also be used. Furthermore, anti-digoxigenin antibodies, anti-dinitrophenyl antibodies, peroxidase-conjugated antibodies that bind to anti-digoxigenin antibodies, etc. can also be used. As the staining substrate, when the labeled antibody is peroxidase for example, diaminobenzidine and the like are used. Proteases, blocking agents, etc. used for pretreatment can also be used.

[0061] When making the target substance detectable using a plurality of antibodies and a staining base material as the staining agent, a plurality of reaction solutions may be used. For example, when using a primary antibody, a secondary antibody, and a staining substrate, a first reaction solution containing the primary antibody, a second reaction solution containing the secondary antibody, and a third reaction solution containing the staining substrate can be used to make the target substance detectable. Also, when using a plurality of reaction solutions, it is sufficient that one of the reaction solutions contains a compound capable of specifically binding to the target substance. In this case, the reaction solution that does not contain the compound capable of specifically binding to the target substance contains a compound capable of binding to the compound capable of specifically binding to this target substance. For example, in the reaction solution containing the primary antibody, the primary antibody corresponds to the compound capable of specifically binding to the target substance. And in the reaction solution containing the secondary antibody, the secondary antibody corresponds to the compound capable of specifically binding to the primary antibody. Furthermore, in the reaction solution containing the staining substrate, the staining substrate corresponds to the compound capable of specifically binding to the secondary antibody.

[0062] In addition, the reaction solution can also be used for multiple staining in which another staining is further performed on a biological sample stained by an immunostaining reaction for composite evaluation. In the case of multiple staining, two or more reaction solutions contain compounds capable of specifically binding to different target substances, respectively.

[0063] (Aqueous medium) The reaction solution preferably contains an aqueous medium. As the aqueous medium, it preferably contains water. Further, as the water, deionized water or ion-exchanged water is preferably used. The content (mass%) of water in the reaction solution is preferably 25.0 mass% or more and 99.0 mass% or less based on the total mass of the reaction solution.

[0064] In addition, the aqueous medium preferably contains a water-soluble organic solvent together with water in order to improve the staining property and shorten the staining time. Here, the water-soluble organic solvent refers to an organic solvent having a solubility in water at 20°C of 200 g / L or more. The content (mass%) of the water-soluble organic solvent is preferably 75.0 mass% or less based on the total mass of the reaction solution.

[0065] Examples of the water-soluble organic solvent include alcohols, (poly)alkylene glycols, glycol ethers, nitrogen-containing compounds, and sulfur-containing compounds.

[0066] In addition, as the aqueous medium, a solution obtained by dissolving a water-soluble organic compound that is solid at 25°C in water can be used. Examples of the water-soluble organic compound that is solid at 25°C include urea and its derivatives, polyethylene glycol having an average molecular weight of 1000 or more, trimethylolpropane, and trimethylolethane.

[0067] (Buffer solution) The reaction solution preferably contains a buffer. By using a buffer, changes in the pH of the reaction solution can be suppressed, and the staining property can be stabilized. Examples of the buffer include phosphate buffer, glycine buffer, Good's buffer, Tris buffer, ammonia buffer, and the like. Also, when the buffer contains an aqueous medium, the aqueous medium is considered as a part of the aqueous medium in the reaction solution.

[0068] (Salt) The reaction solution can contain a salt. By adjusting the concentration of the salt in the reaction solution, the staining conditions can be optimized. Examples of the salt include sodium salts such as sodium chloride, potassium salts such as potassium chloride, and magnesium salts such as magnesium chloride.

[0069] (Other components) In addition, the reaction solution may contain various additives such as defoaming agents, surfactants, pH adjusters, viscosity adjusters, rust preventives, preservatives, antifungal agents, antioxidants, anti-reducing agents, and chelating agents as needed in addition to the above components.

[0070] <Specific examples of a method for preparing a biological sample for analysis and a method for identifying a target substance> Specific examples of a method for preparing a biological sample for analysis and a method for identifying a target substance will be described below.

[0071] Pretreatment may be performed to make the biological sample in a state that is easy to identify. For example, in order to make the biological sample easy to identify, the biological sample may be sectioned thinly. At this time, in order to make it easy to section the biological sample thinly, an embedding agent such as paraffin may be used. Hereinafter, a method for identifying a target substance during immunostaining in a biological sample embedded in paraffin after being sectioned thinly will be taken as an example to explain the method for identifying a target substance.

[0072] In a biological sample embedded in paraffin, the embedding agent is removed prior to identification such as staining to expose the biological sample. As the organic solvent used for removing the embedding agent, xylene, ethanol, etc. can be used. Further, if necessary, treatments such as antigen activation treatment, protease treatment, blocking treatment, etc. may be performed as needed.

[0073] The biological sample with the embedding agent removed is placed on a substrate, and the outline of the biological sample on the substrate is specified. Then, based on the information regarding the outline of the biological sample, a holding part is formed by applying a first liquid composition and a second liquid composition along the outline of the biological sample, respectively.

[0074] After forming the holding part, a first reaction solution containing a primary antibody that binds to the antigen which is the target substance in the biological sample is dispensed, and after a desired period of time has elapsed, a washing treatment is performed. Then, a second reaction solution containing a peroxidase-conjugated secondary antibody is dispensed, a reaction for binding the primary antibody and the secondary antibody is performed, and a washing treatment is performed. Further, a third reaction solution containing diaminobenzidine which is a staining agent is dispensed, after reacting the secondary antibody and the staining agent, a washing treatment is performed. If necessary, counterstaining with hematoxylin solution is performed and then a washing treatment is performed.

[0075] After staining the target substance in this way, the biological sample is confirmed by microscopic observation. If necessary, the specimen may be confirmed after an encapsulation operation.

[0076] The method for preparing a biological sample for analysis and the method for identifying a target substance according to the present invention are not limited to only the immunostaining exemplified above. It can also be applied to staining with dyes such as HE staining, and staining using DNA and RNA such as in situ hybridization. Also, when it is desired to use a combination of methods for identifying a plurality of target substances, a plurality of holding parts can be prepared and used.

[0077] [Dryness Inhibiting Film Forming Device] The preparation device for a biological sample for analysis preferably further has a drying inhibition film forming device (hereinafter also referred to as a "film forming device"), which is a drying inhibition film forming means. The film forming device is a device that forms a drying inhibition film so as to cover the surface of the biological sample to which the reaction solution has been applied in order to inhibit the drying of the reaction solution after the step of applying the reaction solution to the biological sample and binding a compound to the target substance. When a volatile solvent is contained in the reaction solution, it may not be possible to efficiently bring the reaction solution into contact with the target substance due to the volatilization of the solvent. In contrast, it is preferable to form a drying inhibition film using the film forming device after applying the reaction solution to the biological sample, because the drying of the reaction solution can be inhibited.

[0078] The film forming device is not particularly limited as long as it can form a drying inhibition film on the surface of the biological sample to which the reaction solution has been applied. For example, there are liquid application devices such as a spray method, a shower method, a dispenser method, and an inkjet method that can apply a liquid substance as droplets. Further, when the film forming device is a liquid application device, it is possible to adjust the application amount of the liquid substance for forming the drying inhibition film. For example, the application amount of the liquid substance can be adjusted by adjusting the diameter or number of nozzles used when applying the liquid substance, or the pressure applied to the liquid substance. Further, when the liquid application device is a device having an electrical control unit, the application amount of the liquid substance can be adjusted by opening and closing the flow path of the liquid substance for forming the drying inhibition film. The application amount of the liquid substance for forming the drying inhibition film can be controlled based on information such as temperature and pressure, or by an external input.

[0079] In addition, the drying inhibition film is not particularly limited as long as it can inhibit the drying of the reaction solution. For example, the drying inhibition film may be formed of a liquid substance. Examples of the liquid substance used to form the drying inhibition film include long-chain fatty acids, long-chain alcohols, mineral oils, silicone oils, phospholipids, and the like. The liquid substance preferably has a slower evaporation rate than the reaction solution. Specifically, long-chain fatty acids and long-chain alcohols having a large number of carbon atoms in the compound are preferable. Examples of the long-chain fatty acid include oleic acid and stearic acid. Examples of the long-chain alcohol include behenyl alcohol and stearyl alcohol. The liquid substance may be a volatile substance, but in that case, the vapor pressure of the liquid substance is preferably smaller than the vapor pressure of the solvent in the reaction solution. A surfactant may be used together with the liquid substance in order to adjust the surface tension of the liquid substance used to form the drying inhibition film.

[0080] <Target Substance Identification Device> For the contour specifying means, liquid ejection means, and reaction solution applying means included in the target substance identification device, the same ones as those used in the preparation device for the biological sample for analysis can be used. Therefore, the description of the contour specifying means, liquid ejection means, and reaction solution applying means will be omitted. Hereinafter, the detection device, which is the identification means included in the target substance identification device, will be described.

[0081] [Detection Device] The target substance identification device has a detection device for detecting a target substance bound to a compound that can specifically bind to the target substance. When the compound that can specifically bind to the target substance binds to the target substance, the target substance becomes in a detectable state. The detectable target substance may be detected visually or by detection, but in order to quantitatively detect the target substance, it is preferable to use a detection device.

[0082] The detection device is not particularly limited, and known ones can be used. Specifically, an optical microscope, a fluorescence microscope, etc. can be mentioned.

Example

[0083] Hereinafter, the present embodiment will be described in more detail using examples and comparative examples. The present invention is not limited in any way by the following examples as long as the gist thereof is not exceeded.

[0084] Using an identification device provided with a detection device in addition to the preparation device 100 shown in FIG. 1, a biological sample for analysis was prepared and a target substance in the biological sample was identified. As the preparation device 100, a modified inkjet recording device having an inkjet liquid ejection head was used. Instead of the ink cartridge mounted on the inkjet recording device, cartridges containing a reaction solution for identifying a target substance and a liquid composition for forming a holding portion were attached to the carriage, respectively. Thereby, the reaction solution and the liquid composition can be ejected from the inkjet liquid ejection head. In addition, an imaging sensor, which is a contour specifying device for specifying the contour of the biological sample, is provided. These are controlled by a computer through a circuit board in the same manner as a general inkjet printer.

[0085] A slide glass on which a biological sample is fixed is placed on the preparation device 100 shown in FIG. 1, and the biological sample is photographed using the imaging sensor. The photographed information regarding the contour of the biological sample is stored in the computer. Based on this photographed information, an application pattern of the liquid composition and the reaction solution is set. There are two types of application patterns. One is application pattern I for applying the liquid composition to form a holding portion along the contour of the biological sample, and application pattern II for applying the reaction solution inside the formed holding portion (inside the contour of the biological sample) is used.

[0086] These are applied in the set pattern order. Further, as the liquid compositions, a first liquid composition and a second liquid composition are used. The first liquid composition contains a cationic resin, an alkyl ketene dimer, and an aqueous medium, and the second liquid composition contains an anionic resin, an alkyl ketene dimer, and an aqueous medium. By discharging the first liquid composition and the second liquid composition from an inkjet-type liquid ejection head and bringing them into contact on a substrate, the cationic resin and the anionic resin are reacted. Thereby, a holding part (partition wall) was continuously formed so as to cover the periphery of the contour of the biological sample. Further, this holding part has water repellency.

[0087] (Example 1) Example 1 shows a method for preparing a biological sample for analysis in the case of an immunostaining reaction and a method for identifying a target substance.

[0088] As a compound that can specifically bind to the target substance contained in the reaction solution, cancer marker serum HER2 is used.

[0089] A cultured cell (biological sample) having HER2 antigen (target substance) is placed on a slide glass and installed in the identification device. First, the contour of the biological sample is specified by the contour specifying device 101. Then, along the contour of the specified biological sample, the first liquid composition and the second liquid composition are applied in application pattern I and dried to form a holding part (partition wall).

[0090] Next, a first reaction solution containing an anti-HER2 primary antibody is applied inside the holding part in application pattern II, and the anti-HER2 primary antibody is reacted with the HER2 antigen in the biological sample. Then, the slide glass is washed with a buffer solution to separate the excess anti-HER2 primary antibody.

[0091] Next, a second reaction solution containing an anti-HER2 secondary antibody containing an enzyme as a catalytic function for color development is applied inside the holding part in the application pattern II, and the anti-HER2 secondary antibody is reacted with the anti-HER2 primary antibody bound to the HER2 antigen in the biological sample. Then, the slide glass is washed with a buffer solution to separate the excess anti-HER2 secondary antibody.

[0092] Next, a third reaction solution containing a substrate for color development is applied inside the holding part in the pattern II, and the substrate is reacted with the anti-HER2 secondary antibody bound to the HER2 antigen in the biological sample. Then, the slide glass is washed with a buffer solution to separate the unreacted substrate.

[0093] This slide glass is dried and observed with an optical microscope which is a detection device.

[0094] The color density increases according to the abundance of the HER2 antigen, the color development can be quantified, and positive and negative determinations can be carried out according to known color density determination criteria. Also, a fine holding part can be formed along the contour of the biological sample using an inkjet type liquid ejection head, and the application amount of the reaction solution can be reduced.

[0095] (Example 2) Example 2 shows a method for preparing a biological sample for analysis in the case of in situ hybridization and a method for identifying a target substance.

[0096] As a compound capable of specifically binding to the target substance contained in the reaction solution, cancer marker serum HER2 is used.

[0097] Place the cultured cells (biological sample) on a slide glass and heat-denature it at 95°C for 30 minutes so as to unwind the double helix structure of the DNA in the biological sample. Place the slide glass with the cultured cells having the heat-denatured DNA thereon in the identification device. First, the contour of the biological sample is specified by the contour specifying device 101. Then, along the contour of the specified biological sample, the first liquid composition and the second liquid composition are applied with the application pattern I and dried to form a holding part (partition wall).

[0098] Next, a reaction solution containing a HER2 probe labeled with a fluorescent molecule is applied inside the holding part with the application pattern II to react the HER2 probe with the heat-denatured DNA in the biological sample.

[0099] During this period, since water evaporates and the concentration of the reaction solution is likely to change, the reaction solution is applied periodically to suppress this concentration change. Then, the slide glass is washed with a buffer solution to separate the excess probe.

[0100] Dry this slide glass and observe it with a fluorescence microscope which is a detection device.

[0101] The color density increases according to the abundance of the HER2 antigen in the heat-denatured DNA, the color development can be quantified, and the positive and negative determinations can be carried out in light of the known color density determination criteria. Also, a fine holding part can be formed along the contour of the biological sample using an inkjet-type liquid ejection head, and the application amount of the reaction solution can be reduced.

[0102] (Example 3) In Example 3, instead of applying the reaction solution to suppress the change in the concentration of the reaction solution in Example 2, an oil film which is a drying suppression film is formed on the surface of the biological sample to which the reaction solution has been applied by a drying suppression film forming device.

[0103] The color density increases according to the abundance of the HER2 antigen in the heat-denatured DNA, enabling quantification of the color development and allowing for positive and negative determinations in accordance with known color density criteria. Additionally, a fine holding portion can be formed along the contour of the biological sample using an inkjet-type liquid ejection head, reducing the amount of reaction solution to be applied.

Explanation of symbols

[0104] 100 Preparation device 101 Contour identification device 102 Liquid ejection device 103 Reaction solution application device 104 Biological sample 105 Substrate 106 Holding portion 107 Reaction solution

Claims

1. 1. A method for preparing a biological sample for analysis, comprising the steps of: identifying a contour of the biological sample on a substrate; forming a holding portion on the substrate along the contour using an inkjet liquid ejection head for holding at least a portion of a reaction solution containing a compound capable of specifically binding to a target substance in the biological sample inside the contour; applying the reaction solution to the biological sample to obtain the biological sample for analysis; 1. A method for preparing a biological sample for analysis, comprising:

2. 2. The method of preparing a biological sample for analysis of claim 1, wherein said step of identifying said contour comprises optically identifying said contour.

3. 2. The method of claim 1, wherein the retaining portion is formed to contact and conform to the contour.

4. 2. The method of claim 1, wherein the retaining portion is formed continuously around the periphery of the outline.

5. 2. The method for preparing a biological sample for analysis according to claim 1, wherein the holding portion is formed by using a plurality of the liquid ejection heads, ejecting liquid compositions containing different types of constituent materials of the holding portion from the plurality of liquid ejection heads toward the substrate, and causing the liquid compositions to come into contact with each other on the substrate.

6. 6. The method of claim 5, wherein the liquid compositions are a first liquid composition comprising a cationic resin and a second liquid composition comprising an anionic resin.

7. 2. The method of claim 1, wherein the holding portion is a partition wall that divides a space for holding the reaction liquid inside the contour.

8. 8. The method of claim 7, wherein the minimum height of the partition wall from the surface of the substrate is greater than the maximum height of the biological sample from the surface of the substrate.

9. In the step of identifying the contour, the contour is optically identified; In the step of forming the holding portion, the holding portion uses a plurality of the liquid ejection heads, 2. A method for preparing a biological sample for analysis as described in claim 1, wherein liquid compositions containing different types of constituent materials of the holding portion are ejected from multiple liquid ejection heads toward the substrate, and the liquid compositions are brought into contact with each other on the substrate.

10. 2. The method of preparing a biological sample for analysis according to claim 1, wherein the reaction liquid is applied to the biological sample by ejecting it from an inkjet type liquid ejection head toward the biological sample.

11. 2. The method for preparing a biological sample for analysis according to claim 1, further comprising forming a drying-suppression film for suppressing drying of the reaction liquid so as to cover the surface of the biological sample to which the reaction liquid has been applied, after the step of applying the reaction liquid to the biological sample and binding the compound to the target substance.

12. 1. An apparatus for preparing a biological sample for analysis, comprising: a contour identifying means for identifying a contour of the biological sample on a substrate; a liquid ejection means having an inkjet type liquid ejection head that forms a holding portion on the base material along the contour for holding at least a part of a reaction liquid containing a compound capable of specifically binding to a target substance in the biological sample inside the contour; a reaction solution applying means for applying the reaction solution to the biological sample to obtain the biological sample for analysis; 1. An apparatus for preparing a biological sample for analysis, comprising:

13. A method for identifying a target substance in a biological sample, comprising: identifying a contour of the biological sample on a substrate; forming a holding portion on the substrate along the contour using an inkjet liquid ejection head for holding at least a portion of a reaction solution containing a compound capable of specifically binding to a target substance in the biological sample inside the contour; applying the reaction solution to the biological sample; identifying the target substance by detecting the compound bound to the target substance; A method for identifying a target substance, comprising:

14. A target substance identification device for identifying a target substance in a biological sample, comprising: a contour identifying means for identifying a contour of the biological sample on a substrate; a liquid ejection means having an inkjet type liquid ejection head that forms a holding portion on the base material along the contour for holding at least a part of a reaction liquid containing a compound capable of specifically binding to a target substance in the biological sample inside the contour; A reaction solution applying means for applying the reaction solution to the biological sample; A discrimination means for detecting the compound bound to the target substance; A target substance identification device comprising:

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