WO2025126814A1 - Method for specifying measurement region - Google Patents

Abstract

The present invention addresses the problem of providing a very accurate and highly reproducible method for detecting a target substance present in a sample. This problem is solved by a method for determining a measurement region in a measurement of a target substance present in a sample, the method characteristically comprising a specification step for specifying one or a plurality of holding parts into which the target substance and a standard substance have been introduced and sealed thereinto, wherein, in this specification step, a measurement region is determined by specifying a holding part on the basis of the result of detection of the standard substance.

Description

How to identify the measurement area

The present invention relates to a method for identifying a measurement region in a method for measuring a target substance contained in a sample.

In recent years, attention has been focused on a method of diagnosing diseases such as cancer, Alzheimer's, and other genetic disorders, as well as infectious diseases caused by bacteria, by detecting biomarkers (e.g., nucleic acids such as DNA and RNA, and proteins) contained in a patient's blood and other bodily fluids (liquid biopsy).

Methods for measuring biomarkers include quantification methods of biomarkers using digital counting methods, such as digital ELISA, digital PCR, digital invader, etc. The digital counting method is a technique for measuring the concentration of a biomarker by dividing the biomarker into a number of wells, which are minute (e.g., several μm in size) holding portions, and counting the wells containing the biomarker.
The digital counting method involves binding a target substance (subject to be detected) that is a biomarker to particles, sealing a large number of particles in a large number of wells, and then detecting the number of target substances (the number of wells containing the target substance) (Patent Document 1).

Special Publication No. 2013-521500

The objective of the present invention is to provide a method for detecting a target substance contained in a sample with high accuracy and reproducibility.

In order to solve the above problems, the inventors conducted extensive research and discovered that there are areas where substrate solution remains during the process of sealing the well and sealing is not successful, and that signals from the remaining substrate in the unsuccessful sealing areas cause measurement noise and reduce measurement accuracy. They then discovered that measurement accuracy can be improved by using a fluorescent dye with a different fluorescent wavelength from the substrate used to detect the target substance, identifying areas where substrate solution remains from the observation image of the fluorescent dye, and excluding them from the measurement target, and thus arrived at the present invention. The present invention includes the following aspects.

[1]
A method for determining a measurement area in measuring a target substance contained in a sample, comprising:
identifying one or more holding sections into which the target substance and the standard substance are introduced and sealed;
A method according to claim 1, characterized in that in the identifying step, a holding portion is identified based on a result of detecting the standard substance, and a measurement area is determined.
[2]
The result of detecting the standard substance is an image of the standard substance,
The method according to [1], wherein the identifying step is carried out by creating an image consisting of only the sealed holding portion by excluding an area in the acquired image that is larger than the holding portion.
[3]
the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative,
The method according to [1] or [2], wherein the detection of the standard is carried out by optically detecting fluorescence.
[4]
The method according to [3], wherein the standard substance is a substance containing a fluorescent molecule derivative selected from a fluorescein derivative, a rhodamine derivative, a coumarin derivative, and a cyanine derivative.
[5]
The method according to any one of [1] to [4], wherein two or more capture agents are held in one holding portion.
[6]
The method according to any one of [1] to [5], wherein the holding portion is a microhole provided on the substrate.
[7]
A method for measuring a target substance contained in a sample, comprising the steps of:
a capture step of allowing a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier to capture a labeled or unlabeled substance;
a retaining step of introducing the substance captured by the capture agent into one or more retention sections and retaining the substance;
The method includes a sealing step of sealing the holding portion, and a measurement area determination step of performing the method according to any one of [1] to [6] after the sealing step.
[8]
The method according to [7], wherein in the holding step, a standard substance is also introduced into and held in one or more holding sections.
[9]
After the step of determining the measurement area,
The method according to [7] or [8], further comprising a detection step of detecting a labeled substance captured by the capture agent in the retention area identified in the measurement region determination step.
[10]
the labeled substance is a labeled target substance,
The method includes a labeling step of labeling the target substance with a labeling substance before any step before the sealing step, and
The method according to [9], wherein in the detection step, the target substance labeled in the labeling step is detected.
[11]
the labeled substance is a competitor labeled with a labeling substance;
The method according to [9], wherein the competitor is a substance that binds to the capture agent in competition with the target substance.
[12]
The method according to [10] or [11], further comprising a washing step of washing the holding section holding the labeled substance after the later of the labeling step and the holding step and before the sealing step.
[13]
After the labeling step and the capturing step, whichever is later, and before the sealing step,
The method according to any one of [10] to [12], further comprising a washing step of washing the capturing agent that has captured the labeled substance.
[14]
After the holding step and before the detecting step,
A reaction step of introducing a solution containing a substrate capable of reacting with the labeling substance into the holding portion and reacting the solution with the labeling substance,
The method according to any one of [10] to [13], wherein in the detection step, the substance is detected by detecting a reaction product in the reaction step.
[15]
The method according to [14], wherein the labeling substance is an enzyme and the reaction product is an optically detectable substance.
[16]
The method according to claim 15, wherein the enzyme is peroxidase.
[17]
The method according to [16], wherein the degree of polymerization of the peroxidase is 10 or more and 180 or less.

The present invention can eliminate measurement noise caused by residual substrate in areas where sealing has failed, improving measurement reproducibility and measurement accuracy. Therefore, it is expected that the present invention will provide sufficient reproducibility and measurement accuracy even in methods that detect target substances at high sensitivity and in low concentration ranges. The present invention can detect target substances contained in samples with good reproducibility even at high sensitivity and in low concentration ranges, reducing the time and labor required for the series of operations and reducing costs for materials that form the holding part, detection reagents, solutions, etc., and is expected to contribute to reducing the environmental burden.

FIG. 1 is a diagram showing the structure of a well array 100. FIG. 13 is a photograph (substitute for drawing) showing a fluorescent image of FITC-dextran encapsulated in a well array. FIG. 13 is a diagram (photograph in lieu of drawing) showing an example of an observation target holding region (ROI) identified based on the detection results of a standard substance. FIG. 13 is a diagram (photograph in lieu of drawing) showing an example of an observation target holding portion (ROI) identified based on planar coordinates. 1 is a diagram (photograph as a substitute for a drawing) showing the shadow of a capture agent held in a holding portion. Each scale bar represents 10 μm. 13 is a bright-field image (photograph) showing the variation in the amount of capture agent held in each holder, the scale bar representing 30 μm. FIG. 1 shows the results of BNP measurement and a calibration curve in Example 3.

<Terminology of the present invention>
Holding Part The present invention relates to a method for measuring a target substance using one or more holding parts. The "holding part" may be a compartment for isolating a target substance. The purpose of the holding part may be to isolate the target substance in a compartment for detection and/or reaction, or to distribute the target substance to a plurality of discrete reaction volumes when there are a plurality of holding parts. The holding parts may each be an independent container, may be present on a plurality of substrates, or may be present on a single substrate, and are not particularly limited. Preferably, the holding part is present on a single substrate. In other words, the present invention may use a substrate having one or more holding parts.

In the present invention, "retaining (or making a substance be retained)" simply means that the substance is maintained within a certain compartment. In other words, the substance may or may not be fixed within the compartment, and the substance may or may not be bound to any substance fixed within the compartment, and is not limited to a specific embodiment. The compartment in which the substance is maintained, in other words, "retained," may be the above-mentioned retention section. The method of retaining the substance is not particularly limited, and may be, for example, by gravity, magnetic force, centrifugal force, or any other force. The substance may be retained by fixing it within the compartment, by binding to any substance such as a target substance-binding substance fixed within the compartment, or by not fixing or binding to anything at all, and may be retained by any method.

In the present invention, the size of the holding part is not particularly limited as long as it is capable of holding the target substance. The size of the holding part may be, for example, capable of holding two or more target substances. Furthermore, the size of the holding part may be selected arbitrarily depending on the target substance. Examples of the holding part include a recess or through-hole capable of holding two or more target substances, and a surface covered with a material capable of holding two or more target substances. For example, in the case where the target substance is bound to a cell or exosome, the holding part is preferably a recess or through-hole capable of holding multiple cells or exosomes. The size of the holding part may be determined by the volume of the holding part. Specifically, the volume of the holding portion may be, for example, 0.1 pL or more, 1 pL or more, 7 pL or more, 10 pL or more, 15 pL or more, 100 pL or more, 1 nL or more, 10 nL or more, 100 nL or more, 1 μL or more, 10 μL or more, 50 μL or more, 500 μL or more, 1000 μL or more, 1000 μL or less, 500 μL or less, 50 μL or less, 10 μL or less, 1 μL or less, 100 nL or less, 10 nL or less, 1 nL or less, 100 pL or less, 15 pL or less, 10 pL or less, 7.5 pL or less, 1 pL or less, or a compatible combination of these. More specifically, for example, the volume of the holding portion may be 0.1 pL to 1000 μL, 1 pL to 500 μL, 10 pL to 10 μL, 100 pL to 1 μL, 0.1 pL to 100 nL, 0.1 pL to 10 pL, 10 pL to 1 μL, 1 pL to 1 μL, 1 pL to 100 pL, 1 nL to 1 μL, 10 nL to 1 μL, 100 nL to 50 μL, 1 μL to 1000 μL, or 1 μL to 50 μL.

The number of holding parts can be selected arbitrarily depending on the configuration of the holding parts and the end use. As an example, an array of holding parts with one to several billions can be fabricated using various techniques and materials. The number of holding parts may be increased to increase the dynamic range of the concentration measurement of the target substance. The number of holding parts may be, for example, 1 or more, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 45 or more, 90 or more, 100 or more, 150 or more, 190 or more, 200 or more, 500 or more, 1000 or more, 2000 or more, 5000 or more, 10,000 or more, 100,000 or more, 1 million or more, 10 million or more, 100 million or more, 10 billion or more. The number of holding parts may be, for example, 1 to 100 million, 10 to 1 million, 100,000 or less, 10,000 or less, 5,000 or less, 2,000 or less, 1,000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 25 or less, 15 or less, 10 or less, 8 or less, 6 or less, 5 or less, 3 or less, 2 or less, or any compatible combination thereof. The number of holding parts may be, for example, 1 to 10 billion, 10 to 1 million, 1,000 to 100,000, 1 to 200, or 1 to 100.

The arrangement of the retaining parts is not particularly limited, and may be a planar structure or may be arranged three-dimensionally. Also, they may have a regular design or may be randomly distributed. In one preferred embodiment, the arrangement of the retaining parts may be such that the positions of a regular pattern on a planar structure can be specified on a two-dimensional coordinate plane (e.g., an X-Y coordinate plane).

The retaining portion may be formed of a resin material and/or a solid material, may be formed in a liquid, or may be a combination thereof, and is not particularly limited. The resin material and/or solid material is not particularly limited, and as will be understood by those skilled in the art, there may be a wide variety of possible materials. Some examples of the resin material and/or solid material may include one or more materials selected from the following group: polydimethylsiloxane (PDMS), cycloolefin polymer, cycloolefin copolymer, glass and modified or functional glass, acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon (registered trademark), polysaccharides, nylon or nitrocellulose, composite materials, ceramics, plastic resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, optical fiber bundles, and various other polymers. When the retaining portion is formed in a liquid, it is preferable that the sealing liquid is not mixed with the liquid defining the retaining portion and that the retaining portion is stable. Examples of suitable liquids for encapsulating aqueous reactions include, but are not limited to, water-in-oil emulsions, extruded lipid aggregates, stable suspensions of lipids, liquid crystal aggregates, micelles in water, reverse micelles in oil, and suspensions of cells, bacteria, and viruses.

Specifically, the holding portion may be, for example, an array of microwells. Microwells are small depressions in the surface of the support material. That is, the holding portion may be fine holes provided on the substrate. The microwells may be formed as generally known in the art using any technique, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques. As will be appreciated by those skilled in the art, the technique used may be selected based on the composition and shape of the support material. The materials and shapes are as described above.

The holding portion may be formed to suit the means for sealing it. Furthermore, the sealing of the holding portion may be appropriately selected taking into consideration various conditions such as the shape and material of the holding portion, and whether it is solid or liquid. For example, when multiple holding portions are present on a substrate, the holding portions may be sealed in order to fluidically separate each holding portion so that the contents of the holding portion cannot leak out of the holding portion. Specific methods include, but are not limited to, a method in which a hydrophobic solvent such as silicone oil, mineral oil, or fluorine oil is delivered to the surface on which the holding portion is formed, or a method in which a flat plate or film large enough to cover the entire holding portion on the substrate is uniformly brought into contact with the surface on which the holding portion is formed.

Target substance In the present invention, the target substance is not particularly limited. Examples of target substances include small molecules, environmental pollutants, therapeutic molecules, biomolecules, cells, viruses, spores, etc., or combinations thereof. Preferably, the target substance may be a biomolecule. Non-limiting examples of small molecules include organic compounds and inorganic compounds. Non-limiting examples of environmental pollutants include pesticides, insecticides, and toxins. Non-limiting examples of therapeutic molecules include therapeutic drugs, drugs of abuse, and antibodies. Non-limiting examples of biomolecules include proteins, hormones, antibodies, cytokines, nucleic acids, glycans, carbohydrates, lipids, lipid cell membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands, or combinations thereof. Non-limiting examples of proteins include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, etc. Specific examples of proteins include immunoglobulins, hormones, growth factors, cytokines (many of which act as ligands for cell receptors), cancer markers, and the like, including but not limited to BNP, PSA, and TNF-α. Non-limiting examples of cells include prokaryotic cells (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells. Non-limiting examples of viruses include retroviruses, herpes viruses, adenoviruses, and lentiviruses. The target substance may be bound to a cell, exosome, or viral surface.

When the target substance comprises a nucleic acid, the nucleic acid may be captured by a complementary nucleic acid fragment (e.g., an oligonucleotide) and then, optionally, labeled with a binding ligand comprising a different complementary oligonucleotide.

The target substance may also be an enzyme. Non-limiting examples of enzymes include oxidoreductases, transferases, phosphorylases, hydrolases, lyases, isomerases, ligases, and the like. Further examples of enzymes include, but are not limited to, polymerases, cathepsins, calpains, aminotransferases such as AST and ALT, proteases such as caspases, nucleotide cyclases, transferases, lipases, enzymes associated with heart attacks, and the like. When the systems or methods of the invention are used to detect the presence of viral or bacterial agents, suitable target enzymes include viral or bacterial polymerases and other such enzymes including viral or bacterial proteases, and the like.

Competitive Substance In the present invention, the competitive substance is not particularly limited as long as it binds to a capture agent, usually a target substance-binding substance contained in a capture agent, in competition with the target substance.
For example, the target substance may be any of the above-mentioned examples of a small molecule, an environmental pollutant, a therapeutic molecule, a biomolecule, a cell, a virus, a spore, etc., and may be of the same type as the target substance or of a different type.

Sample In the present invention, the sample is not particularly limited. Examples of samples containing a target substance include blood-derived samples such as whole blood, serum, plasma, blood components, blood cells, blood clots, platelets, or fractions thereof, and other body fluid-derived samples such as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymphatic fluid, bone marrow fluid, tissue fluid, saliva, gastric fluid, synovial fluid, pleural effusion, bile, ascites, amniotic fluid, or fractions thereof. Among them, it is preferable to use the above-mentioned blood-derived sample as the body fluid or fraction thereof. The blood-derived sample may be, for example, a sample that has been treated in advance with an anticoagulant such as citric acid, heparin, or EDTA. The sample may also be a buffer solution, and is preferably a buffer solution containing a target substance. The sample of the present invention is not limited to whether or not it actually contains a target substance, and does not exclude, for example, a sample in which a target substance is not detected by a method for measuring or detecting a target substance, including the measurement method of the present invention.

In the present invention, the insoluble carrier is not particularly limited as long as it is a size that can be enclosed in the holding part and is insoluble in the sample. Non-limiting examples include latex particles, silica colloids, magnetic particles, metal colloids, etc. In particular, when the insoluble carrier has magnetism, it is preferable in that it is easy to hold the carrier in the holding part and to separate the carrier by Bound/Free (B/F).

Target substance-binding substance The composition of the target substance-binding substance depends on the composition of the target substance, and any substance may be used. For example, when the target molecule in the target substance is a protein, the target substance-binding substance may include a protein, particularly an antibody or a fragment thereof (e.g., an antigen-binding fragment (Fab), a Fab' fragment, a pepsin fragment, a F(ab')2 fragment, a full-length polyclonal or monoclonal antibody, an antibody-like fragment, etc.), a receptor protein, other proteins such as protein A, protein G, or a small molecule. When the target molecule in the target substance is an enzyme, suitable target substance-binding substances include enzyme substrates and/or enzyme inhibitors. When the target molecule in the target substance is a phosphorylated species, the target substance-binding substance may include a phosphate binder. Furthermore, when the target molecule in the target substance is a single-stranded nucleic acid, the target substance-binding substance may be a complementary nucleic acid. When the target molecule in the desired substance is a nucleic acid-binding protein, the desired substance-binding substance may be a single-stranded or double-stranded nucleic acid, and vice versa, when the target molecule in the desired substance is a single-stranded or double-stranded nucleic acid, the desired substance-binding substance may be a nucleic acid-binding protein.

Pairs of target substances and target substance-binding substances can include, but are not limited to, combinations such as antibodies and antigens, receptors and ligands, proteins and nucleic acids, nucleic acids and nucleic acids, enzymes and their substrates and/or inhibitors, carbohydrates (including glycoproteins and glycolipids) and lectins and/or selectins, proteins and proteins, proteins and small molecules, and small molecules and small molecules.
In addition, a competing substance may also bind to the target substance-binding substance.

The target substance-binding substance may be attached to the surface of the other substance (e.g., the insoluble carrier) via a linkage, functionalization or modification of the target substance-binding substance and/or a binding surface that facilitates attachment of the target substance-binding substance to the surface of the other substance (e.g., the insoluble carrier described above), and the linkage may include any entity. The linkage between the target substance-binding substance and the surface of the other substance may include one or more chemical or physical (e.g., non-specific attachment via van der Waals forces, hydrogen bonds, electrostatic interactions, hydrophobic or hydrophilic interactions, etc.) bonds and/or chemical linkers that provide such bonds. The target substance-binding substance may be attached to the surface of the other substance by any other known mechanism. The target substance-binding substance may preferably include a first portion that binds to the target substance and a second portion that can be used to attach to the binding surface of the other substance.

The surface of the other material may include a protective or protective layer that can reduce or minimize non-specific attachment of non-target binding substances (e.g., targets, competitors, labeled target binding substances) to the binding surface during the assay, which may result in loss of signal or false positive signals during detection. Examples of materials that may be utilized in certain embodiments to form the protective layer include, but are not limited to, polymers such as polyethylene glycol that repel non-specific binding of proteins; naturally occurring proteins with properties that repel non-specific binding of proteins, such as serum, albumin, and casein; surfactants such as sulfobetaines (e.g., zwitterionic surfactants); naturally occurring long chain lipids; and nucleic acids such as salmon sperm DNA. Thus, attachment of target binding substances to the surface of the other material is not limited to non-specific attachment, but may also be specific attachment. Also, any known technique may be used to attach target binding substances to a wide variety of solid surfaces.

A non-limiting embodiment of the present invention may utilize a proteinaceous target binding agent. Again, any technique known in the art may be used to attach the proteinaceous target binding agent to a wide variety of solid surfaces. As used herein, "protein" or "proteinaceous agent" includes proteins, polypeptides, and peptides, including, for example, enzymes and antibodies. A wide variety of techniques are known to add reactive entities to proteins, such as those outlined in U.S. Pat. No. 5,620,850. Attachment of proteins to surfaces is known, see Heller, Acc. Chem. Res. 23:128 (1990), and many other similar references.

In a non-limiting embodiment of the invention, the target binding agent may include a Fab' fragment. The use of a Fab' fragment, as opposed to a whole antibody, may reduce non-specific binding between the target binding agent and the labeled target binding agent. In some cases, the Fc region of the target binding agent may be removed (e.g., proteolytically). In some cases, an enzyme may be used to remove the Fc region (e.g., pepsin, which may generate F(ab')2 fragments, or papain, which may generate Fab fragments). Sometimes, the target binding agent may be attached to the binding surface using an amine, or modified with biotin (e.g., NHS-biotin) to facilitate binding to an avidin or streptavidin coated capture agent surface. The F(ab')2 fragment may be subjected to a chemical reduction treatment (e.g., by exposure to 2-mercaptoethylamine), which in some cases forms a Fab' fragment that generates two thiols. These thiol-generated fragments can then be attached via reaction with a Michael acceptor, such as maleimide. For example, the Fab' fragments can then be treated with a reagent (e.g., maleimide-biotin) to attach at least one biotin entity (i.e., biotinylation) to facilitate attachment to streptavidin-coated surfaces as described above.

The binding between the target substance-binding substance and the target substance or competitor may be non-specific or specific, and is not particularly limited. When the binding between the target substance-binding substance and the target substance or competitor is specific, for example, the target substance-binding substance and the target substance or competitor may be complementary parts of a binding pair. Furthermore, the target substance-binding substance may specifically and directly bind to the target substance or competitor. "Specific binding" may mean that the target substance-binding substance binds to the target substance or competitor with sufficient specificity to distinguish the target substance or competitor from other components or contaminants in the test sample. The target substance-binding substance may be, for example, an antibody that specifically binds to a portion of the target substance or competitor (e.g., an antigen). The antibody may be any antibody that can specifically bind to the target substance or competitor of interest. Suitable antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to as antibody mimetics), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each. As another example, the target substance or competitor may be an antibody, and the target substance-binding substance may be an antibody.

When the target substance or competitor is a biological cell (e.g., a mammalian, avian, reptile, other vertebrate, insect, yeast, bacterial, etc. cell), the target substance binding agent can be a binding agent that has a specific affinity for a cell surface antigen (e.g., a cell surface receptor). For example, the target substance binding agent can be an adhesion molecule receptor or a portion thereof, which can specifically bind to a cell adhesion molecule expressed on the surface of the target cell type. The adhesion molecule receptor can bind to an adhesion molecule on the extracellular surface of the target cell, thereby immobilizing or capturing the cell. When the target substance or competitor is a cell, the target substance binding agent can be fibronectin, which can have specificity for the target substance or competitor, including, for example, neuronal cells.

The capture agent of the present invention comprises an insoluble carrier and a target substance-binding substance immobilized on the carrier. In the present invention, "immobilization" means that substances are directly or indirectly captured, attached, bound, or added to each other on the binding surface.

The molar ratio of the insoluble carrier to the target substance-binding substance contained in the capture agent is not particularly limited, but may be, for example, within the range of 1:1 to 1:10,000,000, 1:100 to 1:1,000,000, 1:10,000 to 1:100,000, 1:50,000 to 1:100,000, 1:1000 to 1:10,000, 1:10 to 1:1000, 1:1 to 1:100, or about 1:1. It is preferable that the capture agent contains one particle of the insoluble carrier and one or more molecules of the target substance-binding substance. In this case, the target substance-binding substance contained in the capture agent may specifically be, for example, one molecule or more, two molecules or more, three molecules or more, five molecules or more, ten molecules or more, 100 molecules or more, 1000 molecules or more, 10,000 molecules or more, 50,000 molecules or more, 100,000 molecules or more, 1,000,000 molecules or more, 10,000,000 molecules or less, 1,000,000 molecules or less, 100,000 molecules or less, 50,000 molecules or less, 10,000 molecules or less, 1000 molecules or less, 100 molecules or less, 30 molecules or less, 10 molecules or less, 5 molecules or less, 3 molecules or less, 2 molecules or less, or any compatible combination thereof. In this case, the target substance-binding substance contained in the capture agent may, more specifically, be, for example, 1 to 100,000 molecules, 100 to 1,000,000 molecules, 10,000 to 100,000 molecules, 50,000 to 100,000 molecules, 1000 to 10,000 molecules, 10 to 1000 molecules, or 1 to 100 molecules.

The capture agent may be mixed with the sample or with the sample and the labeled competitor. The concentration of the capture agent at the time of mixing is not particularly limited, but may be, for example, 1/mL or more, 100/mL or more, 1,000/mL or more, 10,000/mL or more, 100,000/mL or more, 1,000,000/mL or more, 1,000,000/mL or more, 1,000,000,000/mL or less, 10,000,000/mL or less, 1,000,000/mL or less, 100,000/mL or less, 10,000/mL or less, 100,000/mL or less, 10,000/mL or less, 1000/mL or less, 100/mL or less, or a combination thereof that is not contradictory. More specifically, the concentration of the capture agent contained during mixing may be, for example, 1 particle/mL to 1,000,000,000 particles/mL, 100 particles/mL to 100,000,000 particles/mL, 1,000 particles/mL to 10,000,000 particles/mL, 1,000 particles/mL to 10,000,000 particles/mL, or 10,000 particles/mL to 1,000,000 particles/mL.

The capture agent may or may not contain a substance other than the insoluble carrier and the target substance binding substance, and is not particularly limited. When the capture agent contains a substance other than the insoluble carrier and the target substance binding substance, such a substance is not particularly limited and may contain any known substance. Such a substance may be, for example, a substance contained to immobilize the insoluble carrier and the target substance binding substance, a substance contained to stabilize the insoluble carrier and/or the target substance binding substance, a pre-reaction substrate or detection reagent for the labeled target substance binding substance and/or detection reagent described below, or a substance that emits a signal (e.g., a fluorescent dye, etc.).
The size of the capture agent may be determined in consideration of various conditions, such as the ease of collection of the capture agent by magnetic collection, gravitational sedimentation, etc., the binding amount of the target substance-binding substance, the number that can be retained in the micropores, etc.
As described below, it is preferable that two or more capture agents are held in one holding portion from the viewpoint of improving measurement accuracy, and therefore, it is preferable that the capture agent has a size that allows two or more capture agents to be held in the holding portion. Examples of such a size include, but are not limited to, 1 μm or more and 10 μm or less.

Labeled target substance binding substance and pre-reaction substrate or detection reagent of detection reagent A labeled target substance binding substance is a target substance binding substance labeled with a component that can be detected directly or indirectly by any method. A substance that labels a labeled target substance binding substance may be referred to as a "labeling substance of a labeled target substance binding substance". In the present invention, a labeled target substance binding substance may be used instead of a target substance binding substance or may be used for a labeling step. When a labeled target substance binding substance is used for a labeling step, the labeling step may use at least one labeled target substance binding substance. A labeled target substance binding substance may be selected from any suitable molecule, particle, etc. that can bind to a target substance or a competitor and/or bind to another labeled target substance binding substance.

The detection reagent is a reagent that generates a detectable signal by any method. The signal intensity from the detection reagent preferably increases or decreases to reflect the concentration of the target substance or competing substance. The detection reagent may also be a reaction product converted from a pre-reaction substance in any reaction step. The substance before being converted to the detection reagent is also called the pre-reaction substrate of the detection reagent, or simply the "substrate". The detection reagent is also called the reaction product in the reaction step, or simply the "reaction product". The pre-reaction substrate of the detection reagent does not need to generate a detectable signal. The pre-reaction substrate of the detection reagent and/or the detection reagent may be used for the detection step of the target substance or competing substance and/or the quantification step of the target substance.

The labeled target substance-binding substance may include a component that can facilitate detection, either directly or indirectly. The labeled target substance-binding substance may be a substance that facilitates indirect detection, for example, by converting a pre-reacted substrate of a detection reagent into a detection reagent (e.g., an agent to be detected in an assay).

The labeled target substance-binding substance may, for example, contain an enzyme component (e.g., peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, etc.). When the labeled target substance-binding substance contains an enzyme component, a chromogenic substrate may be used as the substrate. For example, when the enzyme component contains peroxidase, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), o-phenylenediamine dihydrochloride (OPD), etc. may be used as the chromogenic substrate. In this case, the method of binding the target substance-binding substance to the enzyme component may be any method. For example, one of the target substance-binding substance and the enzyme component may have biotin, and the other may have a biotin-binding protein. The labeled target substance binding substance may or may not use an additional labeled target substance binding substance along with the first type of labeled target substance binding substance, and the additional labeled target substance binding substance may be one or more types of labeled target substance binding substance different from the first type of labeled target substance binding substance (e.g., a second type of labeled target substance binding substance, etc.).

More than one type of labeled target binding substance may be used. For example, a first type of labeled target binding substance and a second type of labeled target binding substance may be provided, or at least two, at least three, at least four, at least five, at least eight, at least ten, or more types of labeled target binding substances may be provided. When multiple targets or competitors are exposed to multiple types of labeled target binding substances, at least some of the multiple targets or competitors may be bound to at least one of each type of labeled target binding substance. The labeled target binding substances may be selected so that the labeled target binding substances interact with each other in a variety of different ways. For example, a first type of labeled target binding substance may be capable of binding to a target or competitor, and a second type of labeled target binding substance may be capable of binding to a first type of labeled target binding substance. In these cases, the first type of labeled target binding substance may include a first component that serves to bind to the target substance, may include a second component that serves to bind to the second type of labeled target binding substance, or may include a combination thereof. Specifically, for example, the second component may be biotin, and the second type of labeled target binding substance may include an enzyme or an enzyme component that binds to biotin.

As another example, both the first type of labeled target binding substance and the second type of labeled target binding substance may directly bind to the target substance or competitor. Without being bound by theory or any particular mechanism, the association of both the first type and the second type of labeled target binding substance may provide additional specificity and reliability in performing the assay by identifying only those compartments that are determined to contain both the first type of labeled target binding substance and/or the second type of labeled target binding substance (e.g., via direct or indirect detection) as containing the target substance or competitor. By not considering or counting compartments that are found to have only a single type of labeled target binding substance (e.g., only the first type of detection reagent or only the second type of detection reagent) as containing the target substance or competitor, such an assay method may reduce the number of false positives caused by non-specific binding.

Assays for detecting substances The assays for detecting substances can be performed under a variety of experimental conditions and may be performed in any manner, as will be appreciated by those skilled in the art. The substance detected here may be a target substance or a competitor. The reagents used in the assays for detecting substances are not particularly limited and may be selected appropriately depending on the assay. The reagents include salts, neutral proteins, such as albumin, detergents, and other reagents that can be used to promote optimal protein-protein binding and/or reduce non-specific or background interactions. Other reagents that improve the efficiency of the assay may also be used, such as protease inhibitors, nuclease inhibitors, antibacterial agents, and the like. The mixture of components can be added in any order that provides the necessary binding. As is known in the art, various blocking and washing steps may be performed for the assay for detecting substances. It should be noted that any blocking and/or washing step may be performed in the detection step, but is not limited thereto, and may be performed simultaneously with any other step, or before, after, or in all or any step (including the detection step). The washing step may be, for example, a washing step as described below.

Signal In the present invention, the signal is not particularly limited as long as it can be detected by any method. Examples of the signal include fluorescence, visible light, and radiation. Examples of the substance that emits a signal include a wide variety of dyes including fluorescent dyes and compounds containing radioisotopes. The substance that emits a signal may preferably be a fluorescent dye. Specific examples of the fluorescent dye include organic fluorescent dyes and fluorescent proteins. More specifically, examples of the organic fluorescent dye include fluorescein isothiocyanate (FITC), QuantaRed, 4',6-diamidino-2-phenylindole (DAPI), and the Hoechst family (Hoechst 33258, Hoechst 33342, etc.), and examples of the fluorescent protein include green fluorescent protein (GFP), mCherry, etc.

Standard substance The standard substance is not particularly limited, and any substance can be used. The standard substance may be, for example, a substance that emits a signal. The above description of the signal can be used. The standard substance may be mixed with the sample in advance, or may be introduced together with the capture agent, or before or after the capture agent, or may be introduced into the holding section at any timing. The standard substance may be detected by any method. For example, when the standard substance is a substance that emits a signal, the standard substance may be detected by detecting the signal.

It is preferable that the detection of the standard substance does not inhibit the detection of the labeled substance. For example, when a signal is used to detect the standard substance, the signal is preferably different from the signal used to detect the labeled substance. Specifically, when fluorescence is used to detect the labeled substance, and the standard substance is also detected using fluorescence, it is preferable to select a different wavelength of fluorescence to be used to detect the labeled substance and a different wavelength of fluorescence to be used to detect the standard substance. More specifically, for example, when the standard substance is a substance that emits a signal and the signal is fluorescence, i.e., when the standard substance is a substance that emits fluorescence, it is preferable to select a standard substance whose wavelength of fluorescence is different from the wavelength of fluorescence used to detect the labeled substance.

The fluorescent substance may be a fluorescent molecule or a fluorescent molecule derivative.
The fluorescent molecule may be a single fluorescent molecule, or a compound modified with a fluorescent molecule (such as dextran modified with a fluorescent molecule).
Fluorescent molecules or fluorescent molecule derivatives include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, cyanine and its derivatives, coumarin and its derivatives, cascade blue and its derivatives, lucifer yellow and its derivatives, BODIPY and its derivatives, etc. Fluorescein and its derivatives include fluorescein, fluorescein isothiocyanate (FITC), Oregon Green 488, Oregon Green 514, carboxy-fluorescein (FAM), 5'-dichloro-dimethoxy-fluorescein (JOE). Rhodamine and its derivatives include rhodamine, dichlororhodamine (d-rhodamine), carboxytetramethylrhodamine (TAMRA), carboxy-X-rhodamine (ROX), Texas Red, Alexa Fluor 355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Lissamine, and rhodamine green. Cyanine and its derivatives include indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5, Cy7, picogreen, and SYBR. Coumarin and its derivatives include 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue), 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin (Marina Blue). Cascade Blue™ and its derivatives include Cascade Blue, Cascade Blue Acetyl Azide, Cascade Blue Hydrazide. Lucifer Yellow and its derivatives include Lucifer Yellow CH, Lucifer Yellow Ethylenediamine. Examples of BODIPY and its derivatives include BODIPY 493/503, BODIPY R6G, BODIPY TMR, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/655, etc. Other fluorescent molecules include phycoerythrin, LIZ, VIC, NED, PET, Ribogreen, etc.
The fluorescent molecule or fluorescent molecule derivative used as the standard substance is selected from those that do not affect the measurement of the labeled substance when measuring the target substance in the present invention. When a fluorescent molecule is not used to measure the labeled substance, any of the above-mentioned fluorescent molecules or fluorescent molecule derivatives can be used as the standard substance. When a fluorescent molecule is used to measure the labeled substance, the standard substance is selected from the above-mentioned fluorescent molecules or fluorescent molecule derivatives so as not to affect the excitation wavelength and fluorescence wavelength used in the measurement of the labeled substance.

Method of the Invention
In one aspect, the present invention provides a method for producing a composition comprising:
A method for determining a measurement area in measuring a target substance contained in a sample, comprising:
identifying one or more holding sections into which the target substance and the standard substance are introduced and sealed;
The present invention provides a method (hereinafter, also referred to as the determination method of the present invention) characterized in that in the identification step, a holding portion is identified based on a result of detecting the standard substance to determine a measurement region.

In another aspect, the present invention provides a method for measuring a target substance using the above-mentioned method for determining a measurement region (hereinafter, also referred to as the measurement method of the present invention).
A method for measuring a target substance contained in a sample, comprising the steps of:
a capture step of allowing a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier to capture a labeled or unlabeled substance;
a retaining step of introducing the substance captured by the capture agent into one or more retention sections and retaining the substance;
The method further comprises a sealing step of sealing the holding portion, and a measurement area determination step of performing the above-mentioned method for determining a measurement area after these steps.

Capture step The measurement method of the present invention may include a capture step. In the capture step, a capture agent containing an insoluble carrier and a target substance binding substance immobilized on the carrier is used. When the substance to be detected is a target substance, the capture step can be performed by mixing the capture agent with a sample and allowing the target substance to be captured by the capture agent. When the target substance is labeled in the labeling step before the measurement method of the present invention is performed, the labeled target substance is captured, and when the target substance is not labeled in the labeling step before the measurement method of the present invention is performed, the target substance is captured by the capture agent and then labeled in the labeling step, and in either case, the labeled target substance is detected in the detection step. When the substance to be detected is a competing substance, the capture step can be performed by mixing the capture agent with a sample and the labeled competing substance and allowing the labeled competing substance to be captured by the capture agent. The capture step may immobilize the target substance or competing substance on the binding surface of the insoluble carrier via the target substance binding substance.

In the capture step, the choice of the concentration of the capture agent is not particularly limited, but may depend on several competitive factors. For example, from a thermodynamic and kinetic standpoint, it may be advantageous if there is enough capture agent to capture most of the target analyte. As an illustrative example, thermodynamically, 200,000 capture agents in 100 μL, each bound to about 80,000 target binding substances (e.g., antibodies), may correlate with an antibody concentration of about 0.3 nM, and the equilibrium between the antibody and the protein at that concentration may result in a relatively high capture efficiency of the target substance or competitor in some cases (e.g., >70%). Kinetically, it can be estimated that for 200,000 capture agents dispersed in 100 μL, the average distance between the capture agents is about 80 nm.

Labeling step The measurement method of the present invention may further include a labeling step. In the labeling step, a substance is labeled. For example, in the labeling step, a target substance or a competing substance may be labeled with a labeling substance, and the target substance may usually be labeled with a labeled target substance-binding substance. In the measurement method of the present invention, when the target substance is labeled, it is a measurement method by a so-called non-competitive method, and when the competing substance is labeled, it may correspond to a measurement method by a so-called competitive method.
The timing of carrying out the labeling step is not particularly limited as long as it is carried out before the detection step described later. That is, the labeling step may be carried out before any step before the detection step or before the detection step. For example, the labeling step may be carried out before the capture step, after the capture step and before the holding step, or after the holding step and before the detection step. In addition, when the measurement method of the present invention includes a reaction step described later, the labeling step may be carried out before the reaction step, and when the measurement method of the present invention includes a washing step described later, the labeling step may be carried out before the washing step.
When the substance to be labeled is a competitive substance, the labeling step may be omitted, and usually, a competitive substance labeled with a labeling substance is prepared prior to carrying out the measurement method of the present invention, and then subjected to the measurement method of the present invention.

The labeling of the target substance or the competitor may be carried out, for example, by using a labeled target substance binding substance as the target substance binding substance of the capture agent. In this case, the labeling step may be carried out simultaneously with the capture step. The labeling step may also be carried out by directly or indirectly binding the target substance or the competitor to a labeled substance, or by binding the target substance or the competitor to a labeled target substance binding substance. Specifically, for example, the labeling step may be a step of labeling the target substance or the competitor contained in the sample with a labeled target substance binding substance before the retention step, a step of labeling the target substance or the competitor captured by the capture agent with a labeled target substance binding substance when the capture step is carried out, or a step of labeling the retained target substance or the competitor with a labeled target substance binding substance after the retention step. In these cases, the labeling step may also be carried out before the sealing step described below, but is not limited to this.

In the labeling step, the plurality of targets or competitors (which may be immobilized to a capture agent) may be exposed to the plurality of labeled target-binding substances, such that the labeled target-binding substance binds to at least some of the plurality of targets or competitors. In the labeling step, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 97%, more than about 98%, more than about 99%, or more of the targets or competitors may be labeled. Specifically, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 97%, more than about 98%, more than about 99%, or more of the targets or competitors may bind to the labeled target-binding substance.

When employing one or more labeled target substance binding substances to label a target substance or competitor, it may be advantageous to adjust the concentrations appropriately. For example, considering an embodiment involving a target substance or competitor that is a protein, when employing a labeled target substance binding substance in which the detection antibody is labeled with an enzyme (e.g., peroxidase), the concentrations of the detection antibody and enzyme conjugate (e.g., peroxidase) used to label the protein may in some cases be limited or minimized to provide an acceptable background signal. The selection of the concentrations of the detection antibody and enzyme conjugate (e.g., peroxidase) used to label the protein may be a factor in improving or optimizing the performance of the assay method of the present invention.

Washing Step At least one washing step may be performed before and/or after any step of the present invention. Preferably, the washing step may be performed after the later of the labeling step and the holding step and before the sealing step described below. If a substrate is used, the substrate may be introduced into the holding portion after the washing step. The washing step may be selected so as not to significantly change the target substance or the competitor, or, if a capture agent is used, to significantly change the capture agent, and/or to not destroy any specific binding interaction between at least two components of the assay. The washing solution used in the washing step may also be a solution selected to chemically interact with one or more assay components.

For example, when a capture step is performed, the capture agents may be exposed to one or more solutions containing the target substance or competitor, a labeled target substance-binding substance, etc., and then washed. As another example, following immobilization of the target substance or competitor to the multiple capture agents, the multiple capture agents may be subjected to a washing step, so that any target substance and/or competitor that is not specifically immobilized to the capture agent and any labeled target substance-binding substance that is not specifically immobilized to the target substance are removed. As yet another example, after the target substance and standard substance are introduced into the holding section, the holding section may be washed, so that any substance other than the target substance or competitor and standard substance is removed.

Holding Step In the determination method of the present invention, the target substance and the standard substance are introduced into the specified holding portion and sealed, and such introduction may be performed by the following holding step.
In the retention step, the target substance and the standard substance, and when a competing substance is used, the competing substance, are introduced into a plurality of retention sections and retained. When the capture step is performed, the introduction of the target substance and the competing substance into the retention section may be performed by introducing a capture agent that has captured the target substance or the competing substance into one or a plurality of retention sections and retaining the capture agent in the retention section. The introduction into the retention section may be performed, for example, by filling the retention section with a suspension containing a capture agent that has captured the target substance or the competing substance.
When the capture agent is held in the holder, two or more capture agents may be held in one holder, and in such a case, the number of capture agents (target substances or competitive substances captured by the capture agents) to be detected can be increased, which is preferable since it improves the measurement accuracy. In a preferred embodiment, the holders holding two or more capture agents may account for at least 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of all holders (usually all micropores provided in a substrate) used in the measurement method.

When a substrate is used, the substrate may be introduced into the holding section by first introducing a solution containing the substrate into the holding section together with the target substance and competing substance and retaining them therein, or by adding a solution containing the substrate to the holding section and replacing it with the solution in the holding section. When a solution containing the substrate is added to the holding section and replaced with the solution in the holding section, the target substance or competing substance may be retained and then replaced with the liquid in the holding section, or it may be replaced with a liquid containing a capture agent (if a capture agent is used).

Sealing step In the determination method of the present invention, the target substance and the standard substance, as well as the competing substance if a competing substance is used, are introduced into the specified holding portion and sealed, and such sealing may be performed by the sealing step described below.
The measurement method of the present invention includes a sealing step of sealing the holding parts. The sealing step may be performed, for example, to fluidically separate each holding part so that the contents of the holding parts cannot leak out of the holding parts. The method for sealing the holding parts can be the same as the method described above in the section "holding part".

When a substrate is used, it is preferable to introduce the substrate into the holding section before sealing the holding section. The substrate may be introduced into the holding section by mixing a solution containing the substrate with a capture agent beforehand and holding it in the holding section, or by adding a solution containing the substrate to the holding section and replacing it with the solution in the holding section. When a solution containing the substrate is mixed with a capture agent beforehand and holding it in the holding section, the substrate may be introduced into the holding section simultaneously with the holding step. When a solution containing the substrate is added to the holding section and replacing it with the solution in the holding section, it may be replaced with a liquid containing a capture agent after the holding step or washing step.

Reaction step The measurement method of the present invention may further include a reaction step. The reaction step may be carried out, for example, by introducing a solution containing a substrate capable of reacting with the labeling substance into the holding section and reacting with the labeling substance. In the reaction step, the substrate in the holding section (for example, a pre-reaction substrate of the detection reagent) is converted into a reaction product (for example, the detection reagent). The reaction step may be carried out, for example, after the holding step and before the detection step described below. This causes a reaction between the labeled substance captured by the capture agent and the labeling substance that labels the labeled substance.
A certain period of time may be allowed to elapse in order to convert the substrate into a reaction product. The time of the reaction step is not particularly limited, but the reaction time may be adjusted arbitrarily to the extent that the signal presented by the reaction product (e.g., detection reagent) can be detected. The reaction time may be, specifically, for example, 1 second or more, 5 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 3 minutes or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 3 hours or more, 8 hours or more, or 1 day (24 hours) or more, 3 days or less, 1 day (24 hours) or less, 8 hours or less, 3 hours or less, 1 hour or less, 30 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, 1 minute or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, 5 seconds or less, or 3 seconds or less, or a combination thereof that is not contradictory. More specifically, the reaction time may be, for example, 1 second to 8 hours, 1 second to 30 minutes, 1 second to 5 minutes, 1 second to 30 seconds, 1 second to 3 seconds, 30 seconds to 8 hours, 30 seconds to 10 minutes, 30 seconds to 3 minutes, 1 hour to 3 days, 1 hour to 1 day, or 1 hour to 8 hours. In a specific embodiment, when the labeled target substance-binding substance is labeled with an enzyme, the reaction step may be carried out at an optimal temperature for the reaction of the enzyme.

Identification step In the identification step performed in the determination method of the present invention, a sealed holding part is identified. In one embodiment, the sealed holding part is sealed by the sealing step. In the present invention, the identification step is characterized in that the holding part is identified based on the result of detecting the standard substance to determine the measurement region. In the identification step, the sealed holding part may be identified based on the result of detecting the standard substance. The method of detecting the standard substance can be selected from any method depending on the standard substance, and the result of detecting the standard substance may be in any form depending on the combination of the standard substance and the method of detecting it. The sealed holding part is a holding part that is properly sealed, and may be, for example, a holding part that is sealed independently from other holding parts. The method of identifying the sealed holding part may be achieved, for example, by removing a holding part that is not properly sealed (i.e., failed to be sealed). The holding part that is not properly sealed may refer to, for example, a group of holding parts in which two or more holding parts are connected (hereinafter, also referred to as a "connected region"), may be a holding part in which the solution in the holding part is insufficient, and is not limited as long as it is a holding part in an unintended state. The result of detecting the standard substance may be used to identify the holding part that is not properly sealed. Specifically, for example, an image in which the reference substance is detected, for example a fluorescent image in the case where the reference substance emits fluorescence, may be acquired, and for a region larger than the retaining portion, for example a region having a diameter larger than the diameter of the retaining portion, the retaining portion within that region may be excluded as a retaining portion that is not properly sealed, thereby identifying a retaining portion that is properly sealed. That is, the identifying step may be performed by acquiring an image in which the reference substance is detected, and excluding from the image a region larger than the retaining portion as a region including a retaining portion that is not properly sealed, thereby creating an image consisting of only the sealed retaining portion (hereinafter referred to as a "mask image").

As a specific method for removing the retaining parts that have not been properly sealed, the image in which the reference material is detected may be binarized to create a binarized image, and then contour extraction may be performed to extract the retaining parts and the communication regions that have failed to be sealed. Then, the radius (number of pixels) of the minimum circumscribing circle of each extracted region may be calculated, and if the calculated radius is greater than a preset number of pixels, the region may be determined to be a communication region that has failed to be sealed and removed, thereby creating a mask image that includes only the sealed retaining parts. This allows the position of the retaining parts to be identified and the communication regions to be removed at the same time, and a mask image consisting of only the sealed retaining parts can be easily created.
Furthermore, when the identification of the sealed retaining portion and the creation of the mask image are carried out using a bright field image, if a capture agent is present in the retaining portion, the shadow of the capture agent affects the contour extraction, making it difficult to accurately extract the contour of the retaining portion. However, when the identification is carried out using a fluorescent image of a standard substance that emits high intensity fluorescence, the capture agent in the retaining portion does not affect the contour extraction, which is preferable in that the contour of the retaining portion in the image can be accurately extracted.

The detection of the standard substance can be performed by any method, for example, optical detection. Optical detection can be performed by measuring the color, luminescence, or fluorescence from the standard substance, and is not particularly limited as long as the conditions do not interfere with the detection of the labeled target substance or competing substance. In such cases, the standard substance is a substance that contains a fluorescent molecule or a fluorescent molecule derivative, as described above.

Detection step In the detection step, the target substance or the competing substance in the holding part is detected directly or indirectly. The holding part in the detection step may be the holding part identified in the identification step. The detection of the target substance or the competing substance in the holding part may be performed by a detection reagent. The introduction of the detection reagent into the holding part is not particularly limited, but may be indirectly introduced by converting the pre-reaction substrate of the detection reagent into the detection reagent, or may be introduced by adding the detection reagent directly to the holding part, or when a capture agent is used, may be added after the capture agent solution and replaced with the solution in the holding part before being introduced into the holding part. The conversion of the pre-reaction substrate of the detection reagent into the detection reagent may be performed by the reaction step described above, and the introduction of the pre-reaction substrate of the detection reagent into the holding part can be described in the above "holding step" with reference to the introduction of the substrate into the holding part. The detection method is not particularly limited, but examples include optical, thermal, and electrical methods. When the target substance or the competitor is directly detected, for example, the target substance or the competitor generates a directly detectable signal, or a detection antibody labeled with a directly detectable reagent such as a color-developing reagent is used as a labeled target substance-binding substance. When the target substance or the competitor is indirectly detected, for example, the target substance or the competitor is an enzyme, or the target substance or the competitor does not inherently have enzymatic activity and uses an enzyme-labeled labeled target substance-binding substance. Specifically, for example, when the detection reagent is a reagent that shows a signal such as color development, fluorescence, or chemiluminescence and can be optically measured, a microscope and a CCD camera may be used to image multiple holding parts. Based on the signal derived from the detection reagent in each holding part, the holding part in which the capture agent that captured the target substance or the competitor is held may be detected. In addition, when the reaction step is performed, the detection step may be performed after the reaction step, and specifically, for example, the signal derived from the detection reagent may be measured after the reaction step. When the reaction step is performed, the detection of the target substance or the competitor in the detection step may be performed by detecting the reaction product in the reaction step.

Quantification step The measurement method of the present invention may include a quantification step of quantifying the target substance detected in the detection step. In the quantification step, the target substance is usually quantified based on the detection amount of the target substance or the competing substance detected in the detection step. The quantified value of the target substance may be called a "quantitative value". The method of quantifying the target substance is not particularly limited. The method of quantifying the target substance may be, for example, by applying a measured value, such as a signal intensity, using a calibration curve, or may be performed by directly or indirectly counting the number of molecules, or may be performed by mass spectrometry. When the detection target is a competing substance, the quantification may be performed by converting the quantitative value of the competing substance quantified based on the detection amount to the amount of the target substance whose binding to the target substance-binding substance is competitively inhibited.

It is preferable to first quantify the target substance for each holding part. If there are multiple holding parts, the quantitative values for each holding part may then be added together for the multiple holding parts. For example, if the target substance or competing substance is detected in multiple holding parts, the quantitative values for the multiple holding parts may be added together. For example, in each holding part where the target substance or competing substance is detected in the detection process, the target substance may be quantified for each holding part based on the signal intensity of the signal detected directly or indirectly, and the quantitative values may be added together for all holding parts. If the target substance or competing substance is detected in multiple holding parts, the quantitative values for all of the multiple holding parts may be added together, or any holding part may be selected and added together. The quantitative values may be added up, for example, by adding up the quantitative values for all the holding parts in which the target substance or competing substance was detected, or by selecting any number of holding parts, adding up the quantitative values for each of those holding parts, and then calculating the quantitative values for all the holding parts based on the ratio of the number of the selected holding parts to the total number of holding parts (for example, M holding parts may be selected arbitrarily from a total of N holding parts, the quantitative values for each of those M holding parts may be added up, and the sum may be multiplied by N/M to obtain the sum of the overall quantitative values).

In the present invention, any analysis or evaluation may be performed based on the quantitative value in one or more holding parts or the sum of the quantitative values for each holding part. Specifically, for example, any analysis or evaluation may be performed by regarding the quantitative value of the target substance or the sum of the quantitative values for each holding part as the amount of the target substance contained in the sample.

When a capture agent is used, it is preferable that the signal intensity increases or decreases depending on the number or concentration of the target substance or competing substance captured by the capture agent held in the holding section. In the present invention, in the quantification step, it is acceptable for a single holding section to hold multiple target substances or competing substances, and it is also acceptable for a single holding section to hold two or more capture agents that have captured target substances or competing substances.

Reduction of the influence of the capture agent When a capture agent is used, when a signal is acquired to detect a target substance or a competing substance in a state in which the capture agent is contained in the retention part, the intensity of the signal may vary due to the capture agent in the retention part. For example, the signal may be shielded by the insoluble carrier used in the capture agent, resulting in a decrease in signal intensity. Specifically, for example, when magnetic particles are used as the insoluble carrier used in the capture agent and a fluorescent reagent is used as the detection reagent, the fluorescence may be shielded by the magnetic particles, resulting in a decrease in fluorescence intensity. In the case of a measurement system in which the number of capture agents held in the retention part is adjusted to be constant, particularly so that one capture agent is contained, the fluctuation in signal intensity due to the capture agent is assumed to be the same in each retention part, and therefore does not significantly affect the measurement accuracy. On the other hand, when multiple capture agents are held in one retention part or the amount of the capture agent held varies greatly depending on the retention part, the area occupied by the capture agent varies greatly for each retention part, and the fluctuation in signal intensity due to the capture agent is no longer constant for each retention part, and the fluctuation appears in the output measurement result, for example, the quantitative value in the above-mentioned quantification step, which may adversely affect the measurement accuracy. In the present invention, by reducing fluctuations in signal intensity due to capture agents during the measurement process, it is possible to hold multiple capture agents in one holding portion, thereby improving measurement accuracy.

The signal fluctuation caused by the capture agent may be a decrease in the signal, including the disappearance of the signal, or an increase in the signal, including saturation of the signal. The signal fluctuation caused by the capture agent is not particularly limited, and may be characteristic of the combination of the substance contained in the capture agent, particularly the insoluble carrier, and the type of signal. For example, when the capture agent contains opaque particles such as magnetic particles as the insoluble carrier and the signal is any fluorescence, it is considered that the fluorescence intensity may decrease due to the shielding of the fluorescence by the magnetic particles, etc., and therefore the signal fluctuation caused by the capture agent may be a decrease or disappearance of the signal. As another example, when the signal is any fluorescence and the capture agent contains a substance that emits autofluorescence, the signal fluctuation caused by the capture agent may be an increase in the signal.

In the present invention, various measured values such as the detection of a target substance or a competing substance, the acquired signal intensity, and the quantitative value of a target substance may have the influence of the capture agent reduced when a capture agent is used. Reducing the influence of the capture agent is not limited to performing an operation that directly reduces the influence of the capture agent, but may also indicate performing quantification based on a value obtained through an operation that reduces the influence of the capture agent. Specifically, for example, a quantitative value with reduced influence of the capture agent may not only mean that an operation that reduces the influence of the capture agent is performed when quantifying the target substance, but may also mean a value obtained by reducing the influence of the capture agent when detecting the target substance or a competing substance or acquiring a signal, and then quantifying based on such results.

The reduction of the influence of the capture agent is not particularly limited as long as the final output measurement result does not fluctuate due to the influence of the capture agent. The reduction of the influence of the capture agent may be achieved, for example, by detecting the target substance or competing substance in a measurement range excluding the range affected by the capture agent. In other words, a method of providing an area in the holding section where the capture agent is not present when detecting the target substance or competing substance is exemplified. The method of providing an area where the capture agent is not present may be any method, and may be a method of removing the capture agent from the holding section, a method of accumulating the capture agent in a certain area of the holding section, or a method of transferring the reaction solution in the holding section to another space. The method of removing the capture agent from the holding section may be, for example, solubilizing the capture agent, particularly the insoluble carrier contained in the capture agent, or physically removing the capture agent from the holding section by magnetic force or the like. The method of accumulating the capture agent in a certain region of the holding part may be, for example, to adsorb the capture agent to a specific region such as the wall surface or center of the holding part by magnetic force, to bias the capture agent to a specific region by gravity or centrifugal force, or to naturally bias the capture agent by making the bottom surface of the holding part a non-flat shape such as a concave or convex shape. In the method of transferring the reaction solution in the holding part to another space, the solution may be collected and transferred so as not to contain the capture agent, or the capture agent may be removed with a filter during the transfer. When the reaction solution in the holding part is transferred to another space, the other space to which the capture agent is transferred may be considered as a new holding part. The method of providing an area free of capture agents may be performed after the reaction step. By providing an area free of capture agents prior to the detection of the target substance or competing substance in the detection step, the presence of a capture agent that fluctuates the signal intensity of the capture agent, such as fluorescence, can be suppressed during detection of the target substance or competing substance (for example, image acquisition of the target substance or competing substance by imaging, etc.), thereby improving the measurement accuracy.

The reduction of the influence of the capture agent may be achieved by determining the measurement range using a target substance or a competing substance. For example, when detecting a target substance or a competing substance, the fluctuation of the acquired signal intensity due to the capture agent may be reduced. For example, in the process of detecting a target substance or a competing substance and/or quantifying the target substance, only signals that are less affected by the signal fluctuation due to the capture agent are measured. The method of acquiring a signal that is less affected by the capture agent may be any method, and for example, any number or percentage of pixels on the high-luminance side or low-luminance side excluding 100% may be selected from the observation target region (ROI), low-luminance or high-luminance pixels above a certain level may be excluded, or a method of identifying an area where the capture agent exists from a high-luminance image or a low-luminance image and excluding it from the signal measurement target may be used. Specifically, the method of acquiring a signal that is less affected by the capture agent may select pixels on the high-luminance side or low-luminance side at a percentage of the top 70%, top 50%, top 30%, top 20%, top 10%, top 7.5%, or top 5.0% from the ROI. In particular, the pixels on the high brightness side may be selected from the ROI at a ratio of the top 70%, top 50%, top 30%, top 20%, top 10%, top 7.5%, or top 5.0%. For example, when a capture agent casts a shadow on the signal, a high brightness signal (e.g., a pixel on the high brightness side) may be the measurement target. Also, for example, when a capture agent generates a signal, a low brightness signal (e.g., a pixel on the low brightness side) may be the measurement target. When imaging the holding part to measure the signal, the influence of the capture agent may be reduced before imaging or after imaging the holding part. Prior to detecting the target substance or competing substance in the detection step, i.e., acquiring a signal from the holding part, the measurement accuracy can be improved by excluding the area influenced by the capture agent from the ROI (the capture agent casts a shadow and the brightness is reduced) and analyzing it.

The reduction of the influence of the capture agent may also be achieved by determining the measurement range using a standard substance. For example, a standard substance contained in the solution in the holding section may be used to acquire the signal of the standard substance, a fluctuation in the signal of the standard substance due to the capture agent may be detected, and the signal of the target substance or competing substance may be acquired excluding the region in which the fluctuation in the signal of the standard substance was observed. Alternatively, as another example, the signal of both the standard substance and the signal of the target substance or competing substance in the holding section may be acquired, and the quantification step may be performed excluding the signal of the target substance or competing substance in the region in the holding section in which the fluctuation in the signal of the standard substance due to the capture agent was observed. When the measurement range is determined using a standard substance, the standard substance may be introduced into the holding section by any method at any stage up to the determination of the measurement range.

Control of Enzyme Reaction In the quantification step, a signal intensity detected directly or indirectly according to the concentration of the target substance or competing substance enclosed in the holding section may be used. For example, the enzyme reaction may proceed according to the number or concentration of the target substance or competing substance held in the holding section, and this may be reflected as the signal intensity of the detection reagent. In this case, in order to prevent the signal intensity of the detection reagent from being saturated and the concentration information of the target substance or competing substance from being accurately reflected, the enzyme reaction may be controlled so that a reaction progress state that accurately reflects the concentration information of the target substance or competing substance is maintained. A method for controlling an enzyme reaction that can be expected to achieve more accurate detection and quantification by controlling the enzyme reaction may be a method for inhibiting the enzyme reaction in a reaction progress state that accurately reflects the concentration information of the target substance or competing substance.
An example of a method for controlling an enzyme reaction is the addition of an inhibitor of the enzyme reaction. The timing of the addition may be before the reaction step or at any timing during the reaction step. Preferably, the inhibitor may be added at a timing that accurately reflects the concentration information of the target substance or the competing substance in the reaction progress state.

Another control method is, for example, deactivation of the enzyme by heating. The heating method may be any method, and may be a method of heating the entire holding section, or a method of heating only the solution held in the holding section, or, in the case of using a capture agent, a method of heating the capture agent held in the holding section. The heating temperature is not particularly limited, but is preferably a temperature at which the activity of the enzyme decreases. The timing of heating may be before the reaction process or at any timing during the reaction process. Preferably, heating may be performed at a timing that accurately reflects the reaction progress state of the target substance or competing substance concentration information.

Furthermore, another control method is, for example, adjusting the degree of polymerization of the enzyme used for detection. That is, the enzyme used for detection may be an enzyme polymer. The degree of polymerization is not particularly limited, but it is preferable to be able to provide a signal intensity that reflects the number of target substances or competing substances present in the holding section. The degree of polymerization may be adjusted appropriately according to measurement conditions such as the volume of the holding section and the enzyme reaction time. Specifically, for example, when the enzyme is peroxidase, the degree of polymerization of the peroxidase may be 1 or more, 2 or more, 3 or more, 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 70 or more, 80 or more, 100 or more, 150 or more, 200 or more, 300 or more, or 400 or more, or 500 or less, 400 or less, 300 or less, 200 or less, 180 or less, 150 or less, 100 or less, 80 or less, 70 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, 7 or less, 5 or less, 3 or less, or 2 or less, or any compatible combination thereof. More specifically, the degree of polymerization of peroxidase may be, for example, 1 to 500, 5 to 400, 10 to 180, 10 to 30, 15 to 25, 30 to 50, 70 to 100, 80 to 150, 100 to 300, or 300 to 500. An example of a peroxidase polymer is PolyHRP (Fitzgerald).

The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

<Determination of observation target holding part using standard material>
Example 1: Method for identifying the position of an observation target holding portion based on the detection result of a standard substance (1) A picoliter well array 100 was prepared as shown in Fig. 1. Well array 100 is a substrate comprising a microporous substrate 10 having a plurality of holding portions 11 each having a diameter of 30 µm and a depth of 20 µm, each capable of holding a plurality of capture agents, a 1 mm thick spacer 20 having a through portion 21 on the upper surface of the microporous substrate 10, and a top cover substrate 30 having an inlet 31 for introducing and discharging a sample on the upper surface of the spacer, which are closely attached to each other.
(2) A solution containing a standard substance was introduced into the well array 100 and allowed to stand for 1 minute. In this example, 4 mg/mL Fluorescein isothioxyanate-dextran (Sigma-Aldrich) (hereinafter also referred to as "FITC-dextran") was used as the standard substance. After introduction, the solution was removed, and silicone oil (KF96-20CS, Shin-Etsu Chemical Co., Ltd.) was introduced to seal the solution containing the standard substance (hereinafter also referred to as "FITC solution") in the micropores of the well array.
(3) An inverted fluorescence microscope IX71 (Olympus Corporation) was used to obtain a fluorescence image of FITC-dextran in the holding portion in the through-hole 21 (hereinafter also referred to as a "FITC fluorescence image"). A typical example of the obtained FITC fluorescence image of the well array is shown in Figure 2. As shown in Figure 2, there are independent regions where the FITC solution is sealed in each micropore, and a region 40 where multiple wells are connected.
(4) The acquired FITC fluorescent image was analyzed using image processing software created using OpenCV. Based on the fluorescence brightness detected from the FITC fluorescent image, the observation target holding portion in the acquired image was identified. The method of identifying the observation target holding portion is shown in the following (5) to (9).
(5) The FITC fluorescent image was read into the software as a black and white image, and binarized by Otsu's binarization method to create a binarized image. The binarized image was subjected to contour extraction to obtain information on the number of compartments. A minimum circumscribing circle was created for each compartment, and information on the center and radius of the minimum circumscribing circle for each compartment was obtained.

(6) A mask image was created in which pixels in sections with a minimum circumscribing circle radius of 12 pixels or more were set to "0" and pixels in other areas were set to "1."
(7) The black and white data of the FITC fluorescent image created in (5) was multiplied by the mask image created in (6) to create a converted image in which the pixel values of the sections whose minimum circumscribing circle had a radius of 12 pixels or more were converted to “0.”
(8) The converted image created in (7) was subjected to binarization processing using Otsu's binarization method again to recreate the binarized image. The recreated binarized image was subjected to contour extraction to obtain information on the number of partitions. A minimum circumscribing circle and a rotated circumscribing rectangle were created for each partition, and information on the center and radius of the minimum circumscribing circle and the lengths of the long and short sides of the rotated circumscribing rectangle for each partition was obtained.
Among the sections (9) and (8), sections that satisfy the following three conditions were identified as observation object holding sections.
- The radius of the minimum circumscribing circle obtained in (8) is greater than 4 pixels and less than 12 pixels. - The center of the minimum circumscribing circle obtained in (8) is more than 15 pixels away from the outer frame of the image. - The ratio of the long side to the short side of the rotated circumscribing rectangle obtained in (8) (long side/short side) is less than 2. (10) A region of observation (ROI) measuring 20 pixels square was placed for each identified holding part.

Comparative Example 1 Method for Identifying the Position of the Holding Part Based on Plane Coordinates (1) FITC fluorescent images were obtained by the same methods as in Example 1 (1) to (3).
(2) Using image processing software created using OpenCV, the observation target holding portion in the acquired FITC fluorescent image was identified by a general method using planar coordinates. The method for identifying the observation target holding portion is shown in the following (3) and (4).
(3) First, the coordinates of the first ROI were determined. Specifically, the FITC fluorescent image was loaded into the software, and the top left corner of the first ROI was set to the 19th pixel to the right and the 23rd pixel down from the top left corner of the image, and a first ROI of 20 pixels square was created.
(4) Next, the ROIs were arranged in an array. Specifically, starting from the first ROI created in (3), the ROIs were arranged in 43 columns to the right and 32 rows downward, each spaced 31 pixels apart. However, due to misalignment of the substrate position, misalignment of the substrate angle, substrate distortion, etc., there is a possibility that the well positions may shift for each image. In such cases, the position of the first ROI, and the intervals and inclinations when arranging the ROIs in an array were appropriately adjusted, and each ROI was arranged so as to surround each well.

The ROI for Example 1 is shown in Figure 3, and the ROI for Comparative Example 1 is shown in Figure 4. In the method of Comparative Example 1, which identifies the position of the holding part based on planar coordinates, the inside of communication region 60 is also identified as the observation target holding part. On the other hand, it was confirmed that in the method of Example 1, which identifies the position of the holding part based on the detection results of the standard substance, the inside of communication region 50 is not identified as the observation target holding part. The above results show that by using a standard substance, it is possible to exclude the communication region, which is a cause of measurement errors, from the observation target, and to identify an appropriately sealed holding part as the observation target.

Measurements at the observation object holder determined using standard materials
Example 2: Identifying the position of the observation target holder based on the detection results of the standard substance and measuring BNP The procedure was carried out using magnetic particles onto which BNP was immobilized as the target substance and anti-BNP antibodies as a capture agent, and holder 11 of the well array shown in Figure 1 as the holder.
(1) A BNP standard and a biochemical buffer were mixed to prepare a biochemical buffer containing 3.140 pg/mL BNP.
(2) A 5% (w/v) BSA-containing biochemical buffer solution (hereinafter also referred to as "BSA buffer") and magnetic particles with immobilized anti-BNP antibodies were added to a 2 mL tube.
(3) The solution was brought close to a magnet and left for 1 minute, and the solution was then removed. After that, a washing procedure was carried out twice in which a BSA buffer was added to resuspend the magnetic particles.
(4) The magnetic particle solution was stirred by inversion for 10 minutes or more, and then the stirred solution was brought close to a magnet and left for 1 minute. After removing the solution, 50 μL of BSA buffer was added to resuspend the magnetic particles.
(5) 50 μL of the resuspension solution from (4) and 10 μL of the sample containing 3.140 pg/mL BNP prepared in (1) were mixed in a well of a 96-well plate and stirred for 30 minutes.
(6) After stirring, a magnet was brought close to the bottom of the 96-well plate to accumulate the magnetic particles, and then the supernatant was removed and the plate was washed three times with TBS containing 0.05% (v/v) Tween 20 (trade name) (hereinafter also referred to as "washing buffer").
(7) After removing the solution, 50 μL of a BSA buffer containing a biotin-modified anti-BNP antibody was added and stirred for 15 minutes. The supernatant was removed using a magnet in the same manner as in (6), and the mixture was washed three times with a washing buffer.

(8) After removing the washing solution, 50 μL of BSA buffer containing streptavidin poly-HRP20 conjugate (manufactured by Fitzgerald) polyvalently bound to HRP (horseradish peroxidase) was added and stirred for 15 minutes. The supernatant was removed using a magnet in the same manner as in (6), and the plate was washed three times with the washing buffer.
(9) A magnet was placed close to the bottom of a 96-well plate to accumulate the magnetic particles, and the supernatant was removed, and the magnetic particle solution was resuspended by adding a washing buffer and introduced into the picoliter volume well array shown in Fig. 1. The well array 100 used in this example is a substrate comprising a microporous substrate 10 having a plurality of holding portions 11 each having a diameter of 30 µm and a depth of 10 µm, a 1 mm thick spacer 20 having a through portion 21 on the upper surface thereof, and a top cover substrate 30 having an inlet 31 for introducing and discharging a sample on the upper surface thereof, which are closely attached to each other.
(10) A magnet was brought close to the bottom of the well array, and the magnetic particles were accumulated in each well. The solution was then removed, and a fluorescent substrate reaction solution prepared by mixing 4 mg/mL FITC-dextran with the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher) was introduced, and the mixture was allowed to stand for 1 minute.
(11) A magnet was brought close to the bottom of the well array to accumulate the magnetic particles, after which the solution was removed and silicone oil (KF96-20CS) was introduced and allowed to stand for 15 minutes.
(12) The observation object holding portion was identified in a manner similar to that of Example 1 (3) to (9).
(13) In the fluorescent image of the fluorescent substrate, a region of interest (ROI) of 20 pixels square was set so as to surround the outside of each holder to be observed. However, in this embodiment, as shown in FIG. 5, the capture agent in the holder casts a shadow, reducing the fluorescence intensity of the fluorescent substrate. As shown in FIG. 6, the amount of capture agent held in each holder varies greatly, so the size of the capture agent's shadow on each holder varies greatly. In other words, if the fluorescence intensity of the entire ROI is output, it may contain variations in fluorescence intensity due to the influence of the capture agent's shadow. Therefore, 30 pixels (top 7.5%) on the high-brightness side were selected from the 400 pixels constituting each ROI, and the average of the fluorescence intensities of the selected 30 pixels (top 7.5%) was output as the fluorescence intensity of the fluorescent substrate of each holder, thereby reducing the influence of the capture agent's shadow.
(14) Based on the obtained fluorescence intensity of the fluorescent substrate of each retention site, retention sites having a fluorescence intensity equal to or greater than a preset threshold were extracted as retention sites containing a capture agent that had captured the target substance.
(15) For all the holding parts extracted in (14), the fluorescence intensity of the fluorescent substrate in each holding part obtained in (13) was summed up to calculate the integrated value of the BNP measurement. Note that steps (1) to (15) were performed three times, and the average of the integrated values and the measurement variance (CV) (CV = standard deviation of integrated values / average integrated value × 100) were calculated.

Comparative Example 2: Identifying the position of the holding part based on plane coordinates and measuring BNP (1) As the method for identifying the observation object holding part performed in Example 2, the method shown in Comparative Example 1 (2) to (4) was used, but BNP measurement was performed in the same manner as in Example 2, and the average integrated value and measurement variation (CV) of the BNP sample were calculated.

The results of Example 2 and Comparative Example 2 are shown in Table 1.

The measurement variance (CV) of Example 2, in which the position of the holding part was identified based on the detection results of the standard substance, was 13.0%, while the measurement variance (CV) of the comparative example, in which the position of the holding part was identified based on planar coordinates, was 22.7%. From these results, it was confirmed that the method of the present invention, which can exclude the communication area from the observation object holding part, enables more accurate measurements.

Example 3: Identifying the position of the observation target holding part based on the detection results of the standard substance, measuring BNP, and creating a calibration curve (1) A BNP standard and a biochemical buffer solution were mixed to prepare samples containing BNP, as shown in [A] to [E] below.
[A] Biochemical buffer solution not containing BNP [B] Biochemical buffer solution containing 0.126 pg/mL BNP [C] Biochemical buffer solution containing 0.628 pg/mL BNP [D] Biochemical buffer solution containing 3.140 pg/mL BNP [E] Biochemical buffer solution containing 15.700 pg/mL BNP (2) A biochemical buffer solution containing 5% (w/v) BSA (hereinafter also referred to as "BSA buffer") and magnetic particles having anti-BNP antibodies immobilized thereon were added to a 2 mL tube.
(3) The solution was brought close to a magnet and left for 1 minute, and the solution was then removed. After that, a washing procedure was carried out twice in which a BSA buffer was added to resuspend the magnetic particles.
(4) The magnetic particle solution was stirred by inversion for 10 minutes or more, and then the stirred solution was brought close to a magnet and left for 1 minute. After removing the solution, 50 μL of BSA buffer was added to resuspend the magnetic particles.
(5) 50 μL of the resuspension solution of (4) and 10 μL of any of the samples [A] to [E] prepared in (1) were mixed in a well of a 96-well plate and stirred for 30 minutes.
(6) After stirring, a magnet was brought close to the bottom of the 96-well plate to accumulate the magnetic particles, and then the supernatant was removed and the plate was washed three times with TBS containing 0.05% (v/v) Tween 20 (trade name) (hereinafter also referred to as "washing buffer").
(7) After removing the solution, 50 μL of a BSA buffer containing a biotin-modified anti-BNP antibody was added and stirred for 15 minutes. The supernatant was removed using a magnet in the same manner as in (6), and the mixture was washed three times with a washing buffer.

(8) After removing the washing solution, 50 μL of BSA buffer containing streptavidin poly-HRP20 conjugate (HRP (horseradish peroxidase) conjugated to a polyvalent bond was added and stirred for 15 minutes. The supernatant was removed using a magnet in the same manner as in (6), and the plate was washed three times with the washing buffer.
(9) A magnet was placed close to the bottom of a 96-well plate to accumulate the magnetic particles, and the supernatant was removed, and the magnetic particle solution was resuspended by adding a washing buffer and introduced into the picoliter volume well array shown in Fig. 1. The well array 100 used in this example is a substrate comprising a microporous substrate 10 having a plurality of holding portions 11 each having a diameter of 30 µm and a depth of 10 µm, a 1 mm thick spacer 20 having a through portion 21 on the upper surface thereof, and a top cover substrate 30 having an inlet 31 for introducing and discharging a sample on the upper surface thereof, which are closely attached to each other.
(10) A magnet was brought close to the bottom of the well array, and the magnetic particles were accumulated in each well. The solution was then removed, and a fluorescent substrate reaction solution prepared by mixing the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate with 4 mg/mL FITC-dextran was introduced and allowed to stand for 1 minute.
(11) A magnet was brought close to the bottom of the well array to accumulate the magnetic particles, after which the solution was removed and silicone oil (KF96-20CS) was introduced and allowed to stand for 15 minutes.
(12) The average integrated value and the measurement variation (CV) of each BNP sample were calculated in the same manner as in Example 2(12) to (15).
(13) Using the average value of the integrated value of each BNP sample obtained in (12), a calibration curve is made by 4-parameter logistic regression analysis (4PL).In addition, the integrated value that becomes the detection limit and the lower limit of quantification is calculated from the average value and standard deviation of the integrated value of blank sample (sample [A]), and the concentration that becomes the detection limit and the lower limit of quantification is converted using the created calibration curve.The detection limit is the average value of integrated value + 3.3 x standard deviation, and the lower limit of quantification is the average value of integrated value + 10 x standard deviation.

The results for each BNP sample in Example 3 are shown in Table 2, the detection limit and lower limit of quantification calculated from the calibration curve are shown in Table 3, and the measurement results and calibration curve are shown in Figure 7.

The detection limit was 0.006 pg/mL, and the lower limit of quantification was 0.020 pg/mL, confirming that the target substance can be detected with high sensitivity. In addition, the variance (CV) of the measurement values was 5.2% (sample [B]) to 10.8% (sample [C]), and no significant variance in the measurement values was observed. From these results, it was confirmed that the method of identifying the observation target holding portion of the present invention enables the detection of target substances with high sensitivity and high accuracy.

Example 4 The position of the observation target holding part was identified based on the detection result of the standard substance, and BNP of the plasma sample was measured (1) In Example 2 (5), blood samples were measured in the same manner as in Example 2 (2) to (15), except that plasma for which informed consent was obtained was used as the sample containing BNP, and the average integrated value and the measurement variation (CV) were calculated.

Comparative Example 3: Identifying a holding portion based on plane coordinates and measuring BNP in a blood sample (1) In (5) of Example 2, plasma from which informed consent was obtained was used as a sample containing BNP, and in (12) of Example 2, the method shown in (2) to (4) of Comparative Example 1 was used as the method for identifying the observation target holding portion. Except for this, blood sample measurement was performed in the same manner as in (2) to (15) of Example 2, and the average integrated value and measurement variance (CV) of the blood sample were calculated.
The results of Example 4 and Comparative Example 3 are shown in Table 4.

The measurement variance (CV) of Example 4, in which the position of the holding part was determined based on the detection results of the standard substance, was 5.6%, while the measurement variance (CV) of Comparative Example 3, in which the position of the holding part was determined based on planar coordinates, was 14.1%. These results demonstrate that the method of the present invention, which can exclude the communicating region from the observation target holding part, enables more accurate measurements even for plasma samples.

Example 5: Evaluation of the detection sensitivity of the developed method using blood samples (determining the position of the observation target holding part based on the detection results of the standard substance)
(1) BNP was removed from the plasma of healthy subjects from whom informed consent was obtained, and then BNP was added to the plasma to give one of the following concentrations.
[A] 0.000 pg/mL BNP (add biochemical buffer containing no BNP)
[B] 0.025pg/mL BNP
[C]0.126pg/mL BNP
[D]0.628pg/mL BNP
[E]3.140pg/mL BNP
[F]15.700pg/mL BNP
(2) In Example 2(5), each BNP sample was measured in the same manner as in Example 2(2) to (15), except that each of the BNP samples [A] to [F] prepared in (1) was used as a sample containing BNP.
(3) Using the integrated value of each BNP sample obtained in (2), create a calibration curve by 4PL.In addition, calculate the integrated value of detection limit and the lower limit of quantification from the average value and standard deviation of the integrated value of blank sample (sample [A]), respectively, and convert to the concentration of detection limit and the lower limit of quantification using the created calibration curve.The integrated value of detection limit is the average value of integrated value + 3.3 x standard deviation, and the integrated value of quantification lower limit is the average value of integrated value + 10 x standard deviation.
The results of Example 5 are shown in Table 5.

The detection limit was 0.016 pg/mL, and the lower limit of quantification was 0.047 pg/mL, confirming that the target substance could be detected with high sensitivity even in blood samples.

Example 6: Measurement of variability in the developed method using blood samples (determining the position of the observation target holding part based on the detection results of the standard substance)
(1) In Example 2(5), the BNP measurement of each sample was performed in the same manner as in Example 2(2) to (15), except that the BNP-containing samples used in the calibration curve (biochemical buffer solution containing 0, 0.025, 0.126, 0.628, 3.14, and 15.700 pg/mL BNP) and four plasma specimens (specimens A to D) for which informed consent was obtained were used.
(2) Using the calibration curve created from the integrated values of the BNP samples for the calibration curve obtained in (1), the integrated values of each sample were converted into concentrations, and the average BNP concentration and measurement variance (CV) of each sample were calculated.
The results of Example 6 are shown in Table 6.

The BNP concentrations were all low: 1.666 pg/mL for sample A, 0.704 pg/mL for sample B, 0.109 pg/mL for sample C, and 0.175 pg/mL for sample D. The CVs were 9.1% for sample A, 8.7% for sample B, 5.9% for sample C, and 4.8% for sample D. These results demonstrate that the target substance can be measured with high accuracy even in blood samples with low BNP concentrations.

10 Microporous substrate 11 Holding portion 20 Spacer 21 Penetrating portion 30 Upper cover substrate 31 Inlet 40 Region where multiple wells are connected (connected region)
50 Communication region 60 Communication region 100 Well array

Claims (23)

A method for determining a measurement area in measuring a target substance contained in a sample, comprising:
identifying one or more holding sections into which the target substance and the standard substance are introduced and sealed;
A method according to claim 1, characterized in that in the identifying step, a holding portion is identified based on a result of detecting the standard substance, and a measurement area is determined. The result of detecting the standard substance is an image of the standard substance,
The method of claim 1 , wherein the identifying step is performed by creating an image consisting of only the sealed retaining portion by excluding areas in the acquired image that are larger than the retaining portion. the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative,
The method of claim 1 , wherein the detection of the standard is performed by optically detecting fluorescence. The method according to claim 3, wherein the standard substance is a substance containing a fluorescent molecule derivative selected from a fluorescein derivative, a rhodamine derivative, a coumarin derivative, and a cyanine derivative. The method of claim 1, wherein two or more capture agents are held in one holding portion. The method according to claim 5, wherein the holding portion is a microhole provided on the substrate. A method for measuring a target substance contained in a sample, comprising the steps of:
a capture step of allowing a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier to capture a labeled or unlabeled substance;
a retaining step of introducing the substance captured by the capture agent into one or more retention sections and retaining the substance;
A method comprising a sealing step of sealing the holding portion, and a step of determining a measurement area in which the method according to any one of claims 1 to 6 is performed after these steps. The method according to claim 7, wherein in the holding step, a standard substance is also introduced into one or more holding sections and held therein. After the step of determining the measurement area,
The method according to claim 7, further comprising a detection step of detecting a labeled substance captured by the capture agent in the retention portion identified in the measurement region determination step. the labeled substance is a labeled target substance,
The method includes a labeling step of labeling the target substance with a labeling substance before any step before the sealing step, and
The method according to claim 9 , wherein the target substance labeled in the labeling step is detected in the detection step. The method according to claim 10, further comprising a washing step of washing the holding portion holding the labeled substance after the later of the labeling step and the holding step and before the sealing step. After the labeling step and the capturing step, whichever is later, and before the sealing step,
The method according to claim 10, further comprising a washing step of washing the capture agent that has captured the labeled substance. After the holding step and before the detecting step,
A reaction step of introducing a solution containing a substrate capable of reacting with the labeling substance into the holding portion and reacting the solution with the labeling substance,
The method according to claim 10 , wherein in the detection step, the substance is detected by detecting a reaction product in the reaction step. The method of claim 13, wherein the labeling substance is an enzyme and the reaction product is an optically detectable substance. The method of claim 14, wherein the enzyme is a peroxidase. The method according to claim 15, wherein the degree of polymerization of the peroxidase is 10 or more and 180 or less. the labeled substance is a competitor labeled with a labeling substance;
The method according to claim 9 , wherein the competitor is a substance that binds to the capture agent in competition with the target substance. The method according to claim 17, further comprising a washing step of washing the holding portion holding the labeled substance after the later of the labeling step and the holding step and before the sealing step. After the labeling step and the capturing step, whichever is later, and before the sealing step,
The method according to claim 17, further comprising a washing step of washing the capture agent that has captured the labeled substance. After the holding step and before the detecting step,
A reaction step of introducing a solution containing a substrate capable of reacting with the labeling substance into the holding portion and reacting the solution with the labeling substance,
The method according to claim 17 , wherein in the detection step, the detection of the substance is carried out by detecting a reaction product in the reaction step. 21. The method of claim 20, wherein the labeling substance is an enzyme and the reaction product is an optically detectable substance. The method of claim 21, wherein the enzyme is a peroxidase. The method according to claim 22, wherein the degree of polymerization of the peroxidase is 10 or more and 180 or less.

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