Method of forming microdroplets

Abstract

According to one embodiment of the present invention, it is possible to provide a method for treating an aqueous solution / a method for forming microdroplets, the method comprising: providing a substrate / device / flow path having a surface provided with a hydrophobic surface (region) and a region (nanowire region) surrounded by the hydrophobic surface; the nanowire region is provided with a hydrophilic nanowire; introducing an aqueous solution to the surface of the substrate; and isolating the aqueous solution in the nanowire region.

Description

Technical Field

[0001] The present invention relates to a method for forming a microdroplet array. Background Art

[0002] Various microdroplet arrays have been developed in the past. Compared with earlier technologies such as automatic dispensers and fluidic devices, two-dimensional arrayed microdroplets have achieved high operability, high-speed internal reactions, and limited use of reagents, etc. Microdroplet arrays disperse so-called "terminal dilution" or "limiting dilution" samples in a large number of microdroplets and are used for high-sensitivity, high-precision, and high-speed experiments, analysis, diagnosis, etc.

[0003] The application range of two-dimensional microdroplet arrays covers many fields such as biochemistry, chemistry, and medicine. For example, microdroplet arrays have been put into practical use in droplet digital PCR (ddPCR) (non-patent document 1), digital ELISA (non-patent document 2), etc. Quantification can be performed without a standard curve.

[0004] In one example of a microdroplet array, a hydrophobic surface and a hydrophilic surface are prepared on a plane, and droplets are formed on the hydrophilic surface by surface tension. The volume of the droplets is limited by the bottom area and the surface tension, so the possible size is limited, and in particular, the nanometer size cannot be achieved. Furthermore, the surface tension is determined by the physicochemical properties of the hydrophilic surface. Therefore, the droplets are affected by the manufacturing conditions, the shelf life after manufacturing, exposure to the environment, etc. Therefore, it is costly or difficult to keep the chemical properties of the hydrophilic surface constant just before use.

[0005] In another example of a microdroplet array, a plurality of micropores are provided on the surface of a substrate. Micropores are usually formed using microfabrication techniques based on photolithography and etching. The size of the micropores or their precision depends on the level of microfabrication technology. In particular, nanometer size is difficult to process, and even if it can be processed, the cost is high. Furthermore, in order to isolate the solution in the micropores, the solution is first introduced into the surface of the substrate on which the micropores are arranged, and then the solution outside the micropores is discharged to the outside using oil or the like. At this time, if the micropores are small, for example, about 1 μm or less, it is difficult for the solution to enter the micropores. Furthermore, the solution entering the micropores is easily discharged to the outside together with the oil, and is not easy to remain in the micropores.

[0006] Furthermore, problems have been pointed out in each application. In digital ELISA, the primary antibody is usually fixed to the bottom surface of the hole, beads and other supports, so the reaction efficiency with the target substance is low. In addition, it is said that the direction or posture of the spherical target substance such as extracellular vesicles relative to the primary antibody is not fixed, and the reaction efficiency is low. Therefore, the target substance is easily lost due to the washing step.

[0007] In ddPCR, the target molecule, primers, and polymerase are usually mixed first, and the mixed solution is diluted and introduced into the system to form droplets. Therefore, only a portion of the initial solution is used. Therefore, pre-amplification is required when the concentration of the dilute target molecule is small, such as in single molecule measurement.

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent literature 1: “dPCR: A Technology Review”, Phenix-Lan Quan et al., Sensors (Basel). 2018 Apr; 18(4): 1271

[0011] Non-patent document 2: "Mobile platform for rapid sub-picogram-per-milliliter", multiplexed, digital droplet detection of proteins, Venkata Yelleswarapu etal., PNAS, 116 (10) 4489-4495 Summary of the invention

[0012] According to one embodiment of the present invention, a method for forming droplets can be provided. In some embodiments, the method comprises the following steps: providing a substrate, a device and / or a flow path having a surface, wherein the surface is a surface having a hydrophobic surface (region) and a region (nanowire region) surrounded by the hydrophobic surface, wherein the nanowire region has hydrophilic nanowires; introducing an aqueous solution onto the substrate surface; and isolating the aqueous solution from the nanowire region.

[0013] Thereby, for example, the volume of microdroplets can be controlled within a wide size range, and in particular nano-sized droplets can be formed.

[0014] In some embodiments, the method can be further analyzed digitally. For example, the nanowire can be made to capture the target molecule. Therefore, the target molecule captured by the nanowire is not easy to disappear during the washing step. For example, in the case of digital ELISA, the nanowire can first capture the primary antibody, and then the antigen and the secondary antibody can be introduced. In addition, the nanowire can capture the antigen, and then the detection antibody can be introduced. For example, in the case of ddPCR, a solution containing the target molecule can be introduced, concentrated by drying, and the nanowire can capture the target molecule. Furthermore, for example, in the case of ddPCR, the nanowire can capture the target molecule, and then the primer and polymerase are introduced. Thus, by using smaller droplets, such as nanometer-sized droplets, the volume in the droplets can be reduced, the concentration of the nanowires and the capturing substance can be increased, and the low concentration of the target molecule can be measured with high precision by improving the capture efficiency.

[0015] Further aspects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which merely illustrates and describes exemplary embodiments of the present invention. As will be appreciated, the present invention may have other different embodiments, and some of its details may be modified in various obvious respects without departing from the present invention. Therefore, the drawings and descriptions are to be regarded as illustrative in nature and not as limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A flow chart showing a method of forming droplets according to one embodiment.

[0017] Figure 2 A cross-sectional view of a substrate for explaining a method of manufacturing a substrate for forming droplets according to one embodiment is shown.

[0018] Figure 3 A mask layout for developing dot nanowire regions is shown according to one embodiment.

[0019] Figure 4 Top-down SEM images of dot nanowires of two embodiments are shown.

[0020] Figure 5 Top-down SEM images of dot nanowires of two embodiments are shown.

[0021] Figure 6 Side view SEM images of dot nanowires of two embodiments are shown.

[0022] Figure 7 A three-dimensional fluorescence microscope image after the droplet shape is measured according to one embodiment is shown.

[0023] Figure 8 An exploded perspective view of a flow path device according to an embodiment is shown.

[0024] Fig. 9 A cross-sectional view of a substrate for explaining a process of forming droplets according to one embodiment is shown.

[0025] Fig.10 A cross-sectional view of a substrate for explaining a process of capturing a target substance in an aqueous solution according to one embodiment is shown.

[0026] Fig.11 A fluorescence image (A) and a bright field image (B) of a dot nanowire space according to one embodiment are shown.

[0027] Fig.12 Graphs showing the relationship between the intensity and frequency of fluorescence measured in a dot nanowire space according to three examples are shown.

[0028] Fig.13 Schematic diagram illustrating steps of preparation of a device having dot nanowire spaces, capture of EVs using nanowires, and detection of membrane proteins of captured EVs, according to some embodiments.

[0029] Fig.14 An example of the fluorescence intensity distribution of mCherry and TokyoGreen of a negative control and captured fluorescently labeled EVs is shown.

[0030] Fig.15 The expression frequencies of EVCLDN3 and CD9, and TACSTD2 and CD9 in urine derived from HGSOC patients according to one embodiment are shown. DETAILED DESCRIPTION

[0031] Figure 1 A flow chart showing a method for treating an aqueous solution or a method for forming droplets according to an embodiment of the present invention is shown. First, a flow path device (or substrate) having a nanodot space is provided (S101). An aqueous solution is introduced into the flow path device (S102). The introduced aqueous solution is isolated in the nanodot space (to form droplets) (S103).

[0032] In some embodiments, a device or method is provided for isolating a target substance contained in an aqueous solution, isolating a target substance contained in an aqueous solution in a nanodot space and / or capturing it. In some embodiments, a device or method is provided for measuring a target substance isolated in a nanodot space or captured.

[0033] <Aqueous solution>

[0034] In the present invention, the aqueous solution introduced into the flow path can be a body fluid. "Body fluid" refers to a body fluid obtained from a subject or a sample from the body fluid. The body fluid can be blood, serum, plasma, lymph, interstitial fluid (interstitial fluid), intercellular fluid and other tissue fluids without limitation, and can also be body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (spinal fluid), joint fluid (synovial fluid), aqueous humor (aqueous humor). The body fluid can be a digestive fluid such as saliva, gastric juice, bile, pancreatic juice, intestinal juice, and can also be sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, and milk. The body fluid can be an animal's body fluid or a human's body fluid.

[0035] "Body fluid" may be from a subject of body fluid examination. The subject may be an animal. The subject may be a reptile, a mammal, or an amphibian. Mammals may be dogs, cats, cows, horses, sheep, pigs, hamsters, mice, squirrels, and primates such as monkeys, gorillas, chimpanzees, bonobos, and humans. The subject may particularly be a human.

[0036] The aqueous solution can be a body fluid, a liquid derived from a body fluid (a diluent, a treatment solution, etc.). The aqueous solution can also be a liquid that is not a body fluid (derived from a body fluid), can also be an artificially prepared liquid, or can be a mixed solution of a body fluid or an aqueous solution derived from a body fluid and an aqueous solution derived from a body fluid. The aqueous solution can be an aqueous solution used in a sample assay, or can be an aqueous solution used in an assay for correction. For example, the aqueous solution can be a standard solution or a calibration solution. The specimen to be measured can be a sample. The aqueous solution can include physiological buffers such as phosphate buffered saline (PBS) and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES) containing the recovered substance.

[0037] In some embodiments, the aqueous solution may be a solution for implementing biology, chemistry, biochemistry, physics, optics, or other or a combination thereof in the nanodot space. For example, it may be a solution for implementing PCR (polymerase chain reaction).

[0038] <Target substances>

[0039] In some embodiments, the aqueous solution or aqueous liquid (these terms are used interchangeably in this specification) may contain a target substance. The "target substance" used in this specification generally refers to a substance that is captured by the formed droplets (nanowire space), becomes or may become the object of measurement, detection, observation, quantification, calibration, etc. In some embodiments, the target substance may be an inorganic substance or an organic substance. In some embodiments, the target substance may be a biomolecule.

[0040] The biomolecule may be an organelle or a vesicle. The vesicle may be, but is not limited to, a vacuole, a lysosome, a transport vesicle, a secretion vesicle, a gas vesicle, an extracellular matrix vesicle, an extracellular vesicle, etc., or may contain two or more of them. The extracellular vesicle (EV) may be, but is not limited to, an exosome, an exosome, a shed microvesicle, a microvesicle, a membrane particle, a plasma membrane, an apoptotic vesicle, etc. The vesicle may contain nucleic acids.

[0041] The biomolecule may be a cell without limitation, or may contain a cell. The cell may be a red blood cell, a white blood cell, an immune cell, etc. The biomolecule may be a virus, a bacterium, etc. Non-limiting examples of viruses include influenza virus, coronavirus, and dengue virus.

[0042] Biomolecules may be nucleic acids, i.e., deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). RNA may be messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), etc. DNA / RNA may be cell-free DNA / RNA (cfDNA / RNA), intracellular (e.g., intracellular organelle) DNA / RNA, etc. DNA / RNA may be obtained by liquid biopsy. Nucleic acids may have modifications such as methylation. Biomolecules may be proteins.

[0043] In some embodiments, the target substance may be a biomolecule contained in a biomolecule (eg, a cell, a vesicle, etc.).

[0044] <Substrate>

[0045] "Substrate" used in this specification generally refers to any substrate, or the surface of a material formed on a substrate for performing film processing in a manufacturing process. For example, the substrate surface that can be processed includes materials such as silicon (Si), silicon dioxide (SiO2), silicon on insulator (SOI), germanium (Ge), gallium arsenide (GaAs), glass, sapphire, etc. The substrate surface contains semiconductor materials or materials commonly used in semiconductor processes. The substrate surface contains other materials such as metals, metal oxides, metal alloys and other conductive materials according to the purpose. The substrate surface contains polymer materials. On the substrate surface, oxides, lipid components, etc. can exist at the atomic or microscopic level. The substrate includes semiconductor wafers such as silicon wafers, glass wafers, and polymer films, but is not limited to this. The substrate can be exposed to a pretreatment process to grind, etch, reduce, oxidize, hydroxylate, anneal, clean and / or bake the substrate surface. The substrate surface has these materials as basic constituent materials, for example, it can have impurities or surface layers when processed in the atmosphere or in a clean room environment.

[0046] The surface of the substrate may be substantially flat. The surface of the substrate may have a flat surface. The substrate may be bonded to a member defining a flow path to form a flow path that includes the surface of the substrate inside. The flow path space may be sealed in a fluidic manner and may have an inlet (inlet) or an outlet (outlet). The inlet and outlet may be provided separately, or one or the same opening may be used for both the inlet and the outlet. In some embodiments, the surface of the substrate may have a curved surface at least in part.

[0047] <Hydrophobic surface>

[0048] In some embodiments, the substrate surface may be partially or entirely hydrophobic, may be formed of a hydrophobic material, or may be coated with a hydrophobic film. The "hydrophobic film" used in this specification refers to a film formed of a hydrophobic material (hydrophobic material, the same below) or having a hydrophobic material on the surface. The hydrophobic film may contain a fluorine compound or a resin. The hydrophobic film may be a so-called fluororesin film.

[0049] The hydrophobic material may be selected from the group consisting of fluorinated polymers, perfluorinated polymers, silicon polymers, and mixtures thereof. For example, hydrophobic materials include: amorphous fluoropolymers (commercially available examples include the CYTOP (registered trademark) series manufactured by AGC Chemicals having one of the following terminal functional groups. The hydrophobic material has, for example, one of the following terminal functional groups: A type: -COON, M type: -CONH-Si(OR), or S type: -CF3); polytetrafluoroethylene (commercially available examples include TEFLON (registered trademark) manufactured by Chemours); parylene; fluorinated hydrocarbons; fluoroacrylic copolymers (commercially available examples include FLUOROPEL (registered trademark) manufactured by Cytonix); fluorosilanes (for example, trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOTS); perfluorodecyltrichlorosilane (FDTS) etc.); plasma deposited fluorocarbons; polydimethylsiloxane; other siloxanes; hydrophobic hydrocarbons such as 1-heptadecyne; or mixtures thereof.

[0050] The hydrophobic material can be applied to at least a portion or the entire surface of the substrate. The hydrophobic material can be applied to the substrate surface by a dry method or a wet method. For example, the hydrophobic material can be added dropwise to the substrate surface in a solution state and spin-coated. Thereafter, drying, heat treatment, or baking can be performed.

[0051] <Nanowire region>

[0052] The "nanowire region" used in this specification refers to: a surface region on the substrate surface from which nanowires are grown, or nanowires are arranged or one end or part of a nanowire is maintained. When the nanowires extend outward from the surface region of the seed material in a three-dimensional direction, the nanowire region can be defined by the contour formed by projecting the front end of the nanowire from the upper surface to the substrate surface in a vertical direction. A nanowire region can be defined by a circle or other closed curve, a polygon or other closed broken line, or a combination thereof.

[0053] A region of the nanowire is surrounded by a hydrophobic surface. As described below, the nanowire has hydrophilicity at least on its surface.

[0054] A plurality of nanowire regions may be arranged on the substrate surface. The plurality of nanowire regions or the plurality of nanowire spaces on the substrate surface may be arranged regularly, in a geometric shape or in an array, or irregularly, or in a combination of these.

[0055] <Formation of Nanowire Region>

[0056] In some embodiments, the hydrophobic film formed on the surface of the substrate is patterned to form an opening. In some embodiments, the hydrophobic film can be patterned using photolithography. The resist is applied to the surface of the plastic film by spin coating or spray coating, and pre-baking, exposure and development are performed. Thus, the resist is patterned. The hydrophobic film is exposed at the position where the resist is removed. After development, rinsing, post-baking and other treatments can be performed.

[0057] In some implementations, patterning may include a step of etching using a hard mask (eg, a metal mask).

[0058] In some embodiments, the exposed surface of the hydrophobic film is etched. Thus, the hydrophobic film is patterned corresponding to the pattern of the resist. The etching of the hydrophobic film is not particularly limited, and may be, for example, oxygen etching. Thus, the substrate surface is exposed.

[0059] The size, diameter or radius of the opening (nanowire region) can be 100 μm, 70 μm, 50 μm, 30 μm, 20 μm, 10 μm, 7 μm, 5 μm, 3 μm, 2 μm, 1 μm, 700 nm, 500 nm, 300 nm, 200 nm, 100 nm, 70 nm, 50 nm, 30 nm, 20 nm, 10 nm, etc. or less. The limit of the size of the opening (nanowire region) is usually determined by the accuracy of lithography. For example, UV (ultraviolet) lithography, EUV (extreme ultraviolet) lithography, EB (electron beam) lithography, etc. can be used.

[0060] The substrate can have 1000, 10000, 20000, 30000, 50000, 70000, 100000, 200000, 300000, 500000, 700000, 1000000, 2000000, 3000000, 5000000, 7000000, 10000000, 2000000 In some embodiments, the present invention relates to a plurality of nanowire spaces, wherein the plurality of nanowire spaces have a plurality of nanowire spaces, and the plurality of nanowire spaces have a plurality of nanowire spaces. In some embodiments, the plurality of nanowire spaces have a plurality of nanowire spaces.

[0061] The area of ​​the region where multiple nanowires are arranged in the same space is 100 cm 2 、70cm 2 , 50cm 2 、30cm 2 , 20cm 2 , 10cm 2 、9cm 2 、8cm 2 、7cm 2 、6cm 2 , 5cm 2 , 4cm 2 , 3cm 2 , 2cm 2 , 1cm 2 、0.9cm 2 、0.8cm 2 、0.7cm 2 、0.6cm 2 , 0.5cm 2 、0.4cm 2 、0.3cm 2 , 0.2cm 2 , 0.1cm 2 equal value or smaller.

[0062] In the previous methods for forming micro-droplets, such as micropores, it is difficult to "cut off" droplets of about 1 μm or smaller in size. Therefore, it is difficult to integrate micro-droplets. According to an embodiment of the present invention, the nanowire space can be defined by the bottom surface, that is, the nanowire area, and can be integrated. For example, when UV lithography is used, a nanowire area of ​​μm or hundreds of nm in size can be formed, and when an EB drawing device is used, a smaller nanowire area can be formed. Therefore, in theory, it is possible to manufacture a micro-droplet array with an integration level that meets the structural limits of an optical microscope or an image sensor.

[0063] <Coating of seed material>

[0064] In some embodiments, nanowires are formed in openings or nanowire regions provided in the hydrophobic region on the substrate surface. For example, particles or catalysts for growing nanowires can be applied to the surface of a seed material, and the surface of the seed material can be used as a starting point to grow nanowires. In some embodiments, the material can be a material that can be a catalyst for the growth of nanowires or a starting point for the growth.

[0065] In some embodiments, a seed material for nanowire growth may be applied to the exposed substrate surface. The seed material may be a material that can be a catalyst for nanowire growth or a starting point for growth. The seed material may be applied to the entire substrate, i.e., the surface of the resist. A film may be formed that includes the seed material or is substantially composed of the target material.

[0066] <Removal of photoresist>

[0067] In some embodiments, the photoresist may be removed after the seed material is applied. At this time, the object material applied to the surface of the photoresist may be removed together. This process may be referred to as lift-off. During lift-off, the hydrophobic film and the seed material formed by patterning may remain on the substrate surface.

[0068] <Processing of seed materials>

[0069] In some embodiments, the coated object material may be treated. The treatment may be a treatment of the surface of the object material. The treatment may be heating. The heating may include a step of heating the substrate in a heating furnace, a step of irradiating the object material or the substrate with a laser, and the like. The treatment may be oxidation of at least the surface of the object material, or a portion or the entire volume. Examples of oxidation include heating in an oxidizing atmosphere, plasma treatment, and the like. In some embodiments, the seed material may not be pretreated before the nanowires grow. For example, in the case of using a temperature-sensitive hydrophobic membrane, heating treatment may be avoided.

[0070] <Nanowire>

[0071] "Nanowire" as used in this specification refers to a structure whose maximum diameter, minimum diameter, average diameter, and other characteristic sizes in a cross section are nanometer level (nm), subnanometer level, 10 nanometer level, 100 nanometer level, or submicrometer level when no radius, diameter, or characteristic size, or diameter, etc. is specified. The length of a "nanowire" is a dimension specified in the length direction, which can be from nanometer level to 10 nanometer level, 100 nanometer level, or submicrometer level. "Nanowire" refers to a rod-shaped or linear structure having a nanometer-scale cross-sectional shape, diameter, and other dimensions (non-limitingly, for example, a diameter of 1 to several hundred nanometers). In some embodiments, the length of the nanowires described in this specification is about 0.1 nanometer to about 500 nanometers, about 1 nanometer to about 250 nanometers, about 1 nanometer to about 100 nanometers, or about 5 nanometers to about 50 nanometers.

[0072] The length of the nanowire may be, for example, 500 nm, 1 μm, 1.5 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 17 μm, 20 μm, etc., or more. The length of the nanowire may be, for example, less than 1 μm, 1.5 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 17 μm, 20 μm, 50 μm, 100 μm, 200 μm, etc., or less than these values.

[0073] The diameter (or the size in the thickness direction) of the nanowire may be, for example, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 400 nm, 500 nm, etc. or more, without limitation. The diameter (or the size in the thickness direction) of the nanowire may be, for example, less than 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 400 nm, 500 nm, 1 μm, etc., without limitation, or may be less than these values.

[0074] The cross section of the nanowire may be substantially circular, elliptical, regular polygonal, polygonal, or other shapes. The shape of the nanowire may be substantially cylindrical, elliptical, multi-prism, or other shapes. The nanowire may be hollow or a cavity, or may be a structure substantially filled with a material.

[0075] In some embodiments, nanowires can be directly formed on the substrate without the aid of a catalyst layer or without forming a catalyst layer. In some embodiments, the surface of the substrate (inner wall) or the surface of the catalyst layer where the nanowires are formed or grown can be subjected to surface treatments such as activation treatment, hydrophilization treatment, heat treatment, and hydrothermal treatment. The surface treatment can be, for example, plasma treatment, particle (ion, free radical, neutral atom, etc.) beam irradiation, UV, EUV and other light (electromagnetic wave) irradiation, electron beam irradiation, mechanical treatments such as grinding, etc. The surface treatment can be, for example, a treatment to increase the presence of oxygen that combines with the metal to form a Lewis acid. In some embodiments, performing a surface treatment can include performing a plurality of surface treatments. In some embodiments, two, more than two, or a plurality of surface treatments can be performed simultaneously, can be performed in chronological order, and can be performed in combination.

[0076] The growth method of nanowires can be a physical vapor deposition method such as pulsed laser deposition, VLS (Vapor-Liquid-Solid), CVD (Chemical Vapor Deposition), arc discharge, laser evaporation, organic metal vapor phase selective growth, hydrothermal synthesis, reactive ion etching, calcination, melting, sputtering, etc.

[0077] The material of the nanowire may be an inorganic material or an organic material. The nanowire may be a metal, a non-metal, a semiconductor, a mixture or alloy thereof, or an oxide or nitride thereof, or may include these. The material of the nanowire may be a polymer material, or may include a polymer material. The nanowire may be a wire, a whisker, a fiber, or a mixture or a composite thereof.

[0078] The metal used as the material of the nanowire can be, for example, non-limitingly, typical metals (alkali metals: Li, Na, K, Rb, Cs, alkaline earth metals: Ca, Sr, Ba, Ra), magnesium group elements: Be, Mg, Zn, Cd, Hg, aluminum group elements: Al, Ga, In, rare earth elements: Y, La, Ce, Pr, Nd, Sm, Eu, tin group elements: Ti, Zr, Sn, Hf, Pb, Th, iron group elements: Fe, Co, Ni, earth acid elements: V, N b, Ta, chromium group elements: Cr, Mo, W, U, manganese group elements: Mn, Re, precious metals (copper group, monetary metals): Cu, Ag, Au, platinum group elements: Ru, Rh, Pd, Os, Ir, Pt, radioactive decay products based on natural radioactive elements: U and Th: U, Th, Ra, Rn, actinides, transuranium elements: Np, Pu, Am, Cm, Bk, Cf, Es, Fm, Md, No and other elements after uranium, or their alloys, etc. The nanowires may be any of the above metals or alloys, or oxides of the alloys or mixtures, and may contain oxides.

[0079] The nanowire may be formed of one material or a plurality of materials. The surface of the nanowire may be coated with a coating material.

[0080] The material of the nanowire or at least the surface of the nanowire (e.g., coating material) may be, for example, non-limitingly, ZnO, SiO2, Li2O, MgO, Al2O3, CaO, TiO2, Mn2O3, Fe2O3, CoO, NiO, CuO, Ga2O3, SrO, In2O3, SnO2, Sm2O3, and EuO, etc.

[0081] The nanowires may be charged. The nanowires may have a charge opposite to the charge of the recovered or extracted substance. Thus, as a non-limiting example, charged biomolecules such as extracellular vesicles and nucleic acids may be efficiently attracted or adsorbed.

[0082] <Seed material>

[0083] In some embodiments, nanowires can be grown with the help of a catalyst layer, an adhesive layer, a growth nucleus, and a seed material (layer) (In this specification, these terms are used interchangeably. They are sometimes referred to as "seed materials" below.) A "layer" can be a thin film. A "layer" can be a continuous film. A "layer" can also be discontinuous. A "layer" is a continuous film, and the film can have holes. A "layer" can also be a plurality of thin films separated from each other. A "layer" can be an island or contain islands. A "layer" can be a particle or contain particles.

[0084] The catalyst layer, bonding layer, growth nucleus or seed material layer may be a metal, an alloy, a non-metal, a semiconductor, an oxide, a nitride, etc. thereof, or a mixture thereof. The metal may be, without limitation, a typical metal (alkali metal: Li, Na, K, Rb, Cs, alkaline earth metal: Ca, Sr, Ba, Ra), magnesium group elements: Be, Mg, Zn, Cd, Hg, aluminum group elements: Al, Ga, In, rare earth elements: Y, La, Ce, Pr, Nd, Sm, Eu, tin group elements: Ti, Zr, Sn, Hf, Pb, Th, iron group elements: Fe, Co, Ni, earth acid elements: V, Nb, Ta, chromium group elements: Cr, Mo, W, U, manganese group elements: Mn, Re, precious metals (copper group, monetary metals): Cu, Ag, Au, platinum group elements: Ru, Rh, Pd, Os, Ir, Pt, radioactive decay products based on natural radioactive elements: U and Th: U, Th, Ra, Rn, actinide elements, transuranium elements: Np, Pu, Am, Cm, Bk, Cf, Es, Fm, Md, No and other elements after uranium. The oxide may be any one of them or an oxide of an alloy.

[0085] The particles or seed layer used to form the nanowires may be, for example, Cr, ZnO, CrO, etc. Nanowires using ZnO can be grown on the surface of these seed materials using a hydrothermal synthesis method. In one example, ZnO is first coated on the surface of a substrate (the surface of the target material). Next, the substrate is immersed in a mixture of zinc nitrate hexahydrate (Zn(NO3)2·6H2O), hexamethylenetetramine (C6H 12 N4) is dissolved in a precursor solution formed by deionized water. Thus, ZnO nanowires can be grown on the surface of the seed material.

[0086] The seed material of the nanowire may be formed of a material different from the nanowire. The seed material of the nanowire may be formed of a material substantially the same as the substrate material. The seed material of the nanowire may be, for example, a surface having a concave-convex structure. The seed material of the nanowire may be, for example, a surface having chemically different properties in different parts. A mechanically, structurally or chemically different (mottled) surface is sometimes more likely to become a seed material for a nanowire in some parts than in other parts.

[0087] In some embodiments, a seed material may not be formed. For example, nanowires may be grown directly on the substrate surface, or on the surface of another layer (not a seed material). For example, a concave-convex surface may be formed by photolithography and dry / wet etching. For example, a mechanically, structurally or chemically different (mottled) surface may be formed by irradiating ions, neutral atoms, plasma, etc.

[0088] <Dot Nanowire Space>

[0089] The nanowires are arranged in a direction perpendicular to or inclined to the substrate surface, i.e., three-dimensionally. Therefore, it can define a substantial space for holding an aqueous solution. In this specification, the space can be renamed as nanowire space, point-shaped nanowire space, point (spot) nanowire space, nanodot space, point space, dot (dot) nanowire space. The nanowire space specifies the volume of the droplets formed therein. In this specification, the nanowires constituting the point nanowire space can be renamed as point nanowires, dot nanowires, etc.

[0090] The spacing and bottom area of ​​the dot nanowire region can be delineated by photolithography. On the other hand, the three-dimensional dimensions of the dot nanowire space or its height, width, etc. can be controlled by the growth conditions of the nanowire. The growth conditions include, for example, the concentration of the growth solution, the growth time, etc.

[0091] In some embodiments, the volume of the point nanowire space can be defined by the bottom surface and the space extended by the nanowires. The three-dimensional shape of the point nanowire space can be approximated as a cylinder, or a part of a sphere or ellipsoid, for example. The space extended by the nanowires can be measured, for example, using electron microscope imaging (such as scanning electron microscopy (SEM)) and optical microscopy. In some embodiments, the volume of the point nanowire space can be defined by the volume of the aqueous solution captured by each point nanowire space by its nanowires, either actually or by simulation. For example, for the volume of a droplet of an aqueous solution, its contour can be photographed using optical microscopy. For example, a fluorescent substance can be mixed into an aqueous solution, and the range of fluorescence can be defined as the volume of the point nanowire space using fluorescence microscopy.

[0092] <Method for forming nanowire space: Example 1>

[0093] Figure 2 A process for forming a dot nanowire space according to one embodiment is shown.

[0094] First, a substrate 101 is provided. The substrate 101 may be a semiconductor substrate such as Si or SiO2. Alternatively, the substrate 101 may be a polymer substrate. A hydrophobic film 102 ( Figure 2 A). Figure 2 The hydrophobic film 102 is not limited thereto, and may be formed using other materials and methods.

[0095] A photoresist film 103 is formed on the hydrophobic film 102 ( Figure 2 B).

[0096] The photoresist 103 is developed using a mask prepared in such a way as to open the position where the nanowire region is to be formed. Thus, the Cytop film (hydrophobic film) 102 is exposed at the point (opening) 103A opened by the development ( Figure 2 C).

[0097] An example of a mask is shown in Figure 3 The mask 200 used in this embodiment defines 9 sections (flow path spaces), each section having an area of ​​about 2 cm square ( Figure 3 A). Each segment 201 is surrounded and delimited by a pointless contour portion, and corresponds to a flow path device. In each segment, a plurality of points 202 arranged at the vertices of a plurality of triangles with a side of 6 μm are opened ( Figure 3 B). The distances between points in the figure are shown in mm. The mask 200 has a 1.28×10 7 That is, the integration density is 3,200,000 dots / cm 2 .

[0098] Then, the seed material 105 for nanowire growth is sputter-deposited on the entire surface of the substrate. Figure 2 D). Figure 2 ZnO is evaporated in.

[0099] Then, the photoresist 104 ( Figure 2 E) The removal of the photoresist 104 may be a so-called lift-off. As a result, the seed material (ZnO) 105A remains on the point 105A.

[0100] Then, the nanowire 106 is grown on the surface of the seed material 105 formed at the point 105A by a wet process ( Figure 2 F). Nanowires 106 are grown starting from seed material (ZnO) 105. On the hydrophobic film 102, nanowires do not grow. Thus, nanowires 106 are formed only on the dot region 105A. Therefore, a plurality of dot nanowire regions or spaces can be formed. Generally, nanowires 106 are hydrophilic and are surrounded by the hydrophobic region 102A of the hydrophobic film 102.

[0101] <Control of Nanowire Growth: Example>

[0102] The effect of the concentration of the growth solution on the nanowire growth was investigated. Figure 4 Figure 2 shows SEM images of nanowires grown in the same planar shape using two different growth solution concentrations. The growth solution contains zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and hexamethylenetetramine (C6H 12 N4). In this embodiment, the concentration is 20mM ( Figure 4 A), 40mM( Figure 4 B). Growth time is 2 hours.

[0103] At 20 mM, nanowires were formed thinly ( Figure 4 A). On the other hand, in the case of 40 mM, thicker nanowires were formed compared to 20 mM ( Figure 4 B). Thus, zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and hexamethylenetetramine (C6H 12 The concentration of N4 has an impact on the size (thickness and length, etc.) of the nanowires.

[0104] Figure 5 and Figure 6 ] Figure 2 shows SEM images of nanowires grown in the same planar shape at the same growth solution concentration (30 mM) for different growth times (1 hour, 2 hours). Figure 5 The top views are shown when the growth time is 1 hour (A) and 2 hours (B). Figure 6 The side views are shown when the growth time is 1 hour (A) and 2 hours (B).

[0105] When the growth time was 1 hour, about 150 to about 220 nanowires were formed at each point. The size of the nanowires was 0.094±0.014μm in diameter and 0.88±0.11μm in length. When the growth time was 2 hours, approximately the same number of nanowires were formed at each point. The size of the nanowires was larger in diameter and length than in the case of 1 hour.

[0106] When the growth time is 1 hour, the shape of the nanowire space can be approximated by a hemisphere. The size of the hemisphere is 3.66 μm in diameter and 3.22 μm in height. The volume of the hemisphere is calculated to be V = 1 / 2 × 4 / 3π × 1.83 μm × 1.83 μm × 3.22 μm = 22.6 fL.

[0107] Specifically, the volume of the captured aqueous solution is calculated by subtracting the volume occupied by the nanowires from the space. For example, in the case of the above embodiment, the size of the nanowires is 0.094 μm in diameter and 0.882 μm in length, so the volume is 0.0058 fL. Each nanowire space has about 200 nanowires, so the volume occupied by the nanowires in each nanowire space is calculated to be 1.2 fL. The volume of the aqueous solution captured in the nanowire space is thus calculated to be about 22.6 fL-1.2 fL=21.4 fL.

[0108] In this way, the volume of the femtosecond-sized nanowire space (also referred to as reaction space, reaction field, etc.) can be appropriately adjusted by parameters such as the length of the nanowires.

[0109] The complex aqueous solution was isolated in the same dot nanowire space as above. It was photographed using a fluorescence microscope (Leica, TCS SP5STED). Figure 7 It was thus confirmed that the volume in which the aqueous solution existed, that is, the shape of the droplet could be roughly approximated by a hemisphere and was substantially the same as the volume defined by the shape of the nanowire.

[0110] <Flow path devices>

[0111] In some embodiments, the substrate surface may be bonded to an upper substrate and arranged in a closed space. The substrate surface may be arranged in a flow path or a flow path device. The fluid device may have an inlet and an outlet for a liquid.

[0112] Figure 8 , a flow path device 300 according to an embodiment is shown. The flow path device 300 is manufactured by bonding a first substrate 301 and a second substrate 351 to each other. The first substrate 301 has a substrate surface 302 having a hydrophobic surface (not shown) and a plurality of nanowire regions (not shown) surrounded by the substrate surface 302. The first substrate 301 has a bonding surface 303 at its contour portion, surrounding the substrate surface 302. The second substrate 351 has a recess (recessed space, flow path defining space) 352 arranged opposite to the substrate surface 302 of the first substrate 301. The second substrate 351 has a bonding surface 353 at its contour portion, and is configured to be closely bonded to the bonding surface 303 of the contour portion of the first substrate 301. The second substrate 351 has a solution inlet 353 that is provided through the surface (upper surface of the flow path device 300) on the opposite side from the recessed space 352, and an outlet 354 that is formed in a manner that is open on one side of the flow path device 300 on the opposite side of the surface direction of the recessed space relative to the solution inlet 353.

[0113] Thus, the device 300 formed by bonding the first substrate 301 and the second substrate 351 has a concave space (flow path space) 352 defined by the substrate surface 302 inside. The hydrophobic surface and the plurality of nanowire regions surrounded by the hydrophobic surface are arranged on the substrate surface 302, that is, arranged inside the concave space (flow path space) 352. A solution is introduced into the flow path space 352 from the inlet 353. By introducing, for example, air or oil from the inlet 353 or bringing a water-absorbing material into contact with the outlet 354, the aqueous solution located inside the flow path space 352 and outside the nanowire space can be discharged from the outlet 354. As a result, the aqueous solution can be isolated in the nanowire space.

[0114] The size and dimensions of the flow path device are not particularly limited. As an example, it can be constructed as follows. The first substrate can be a SiO2 substrate, and the second substrate can be a COP (cycloolefin polymer) substrate. Figure 3 It can also be 2cm×2cm. 100 million, 20 million, etc. points can be formed on the surface of the substrate. Figure 3 Similarly, 1.28×10 7 The height of the flow path space can be 50 μm to 10 μm. The size of the inlet 353 can be circular with a diameter of 1 mm or 1.5 mm. The outlet 354 can be 2 cm wide in the substrate surface direction and 50 μm to 10 μm high.

[0115] In some embodiments, the cover (second substrate) may have multiple internal spaces and multiple frames (not shown) surrounding the multiple internal spaces. The cover having multiple internal spaces may be closely attached to multiple target material pattern portions and / or microstructures, and the combined body of the substrate and the cover may be cut or separated according to each internal space.

[0116] In some embodiments, the cover (second substrate) may be transparent, so that light can be injected or extracted, and the droplets can be observed optically or fluorescently.

[0117] In some embodiments, the surface of the cover (second substrate) on at least the flow path space side may be formed of a hydrophobic material. Thus, for example, when the introduced aqueous solution is discharged, the situation where the aqueous solution remains on the surface of the cover in the form of droplets can be avoided or reduced. The hydrophobic material may be a polymer material. For example, the material may be a cycloolefin polymer (COP).

[0118] The "flow path" used in the present invention generally refers to a space for accommodating a fluid. The flow path may also be used interchangeably with terms such as a fluid device, a fluid chamber, and a fluid container. The flow path may have an inlet (inlet) for introducing a fluid therein and / or an outlet (outlet) for discharging a fluid therefrom. A fluid may flow in the flow path, may be introduced into the flow path, and may substantially stop.

[0119] In some embodiments, a so-called chaotic mixer or a structure that causes the fluid flowing in the flow path to flow nonlinearly and / or three-dimensionally may be disposed in the flow path. Such a structure may, for example, have a height difference in the flow path, a change in cross-sectional area, a change in the direction of the flow path, etc.

[0120] A chaotic mixer having herringbone-shaped concave and convex can be arranged on the inner wall surface (surface of substrate or surface of cover) of the flow path. Thus, the nonlinear flow of the fluid can be promoted. Thus, more object substances in the solution can be guided to the microstructure (such as nanowire) arranged on the inner wall surface of multiple inner walls or curved surfaces.

[0121] The fluidic device can detect, measure, analyze, change, process and / or capture substances contained in a fluid introduced into a flow path. The fluidic device can have nanowires. The fluidic device can use nanowires to capture substances contained in a fluid. For example, a fluidic device can capture biomolecules in a body fluid by using nanowires disposed in its flow path.

[0122] <Isolation of droplets>

[0123] In some embodiments, an aqueous solution may be introduced to the substrate surface and removed from the hydrophobic surface, thereby isolating the aqueous solution within the dot nanowire space.

[0124] <Isolation using water-absorbent materials>

[0125] In some embodiments, the water-absorbing material can absorb the aqueous solution located on the hydrophobic surface, thereby removing the aqueous solution from the hydrophobic surface. As a result, for example, the aqueous solution can be isolated in the dot nanowire space. The water-absorbing material contacts a portion of the aqueous solution located on the substrate surface and absorbs the aqueous solution. Thus, the aqueous solution gradually extending to the entire substrate is absorbed into the water-absorbing material in sequence. At this time, it is easy to remove the aqueous solution located in the hydrophobic area. On the other hand, the aqueous solution located in the hydrophilic nanowire area remains in its position. Thus, the aqueous solution is isolated from the nanowire area and remains.

[0126] use Fig. 9 A method for isolating an aqueous solution within a dot nanowire space or forming microdroplets according to one embodiment is described.

[0127] like Fig. 9 As shown in A, a flow path device 400 is provided. The flow path device 400 has a flow path space 452 formed inside by bonding a first substrate 401 and a second substrate 451 to each other. The first substrate 401 has a hydrophobic film 402 on its entire surface. A plurality of nanowire regions 405A on which a plurality of nanowires 406 are arranged are arranged. These are arranged inside the flow path space 452. The second substrate 451 has an inlet 453 and an outlet 454 for a liquid. An aqueous solution 471 is introduced into the flow path space 452 from the inlet 453 and fills the flow path space 452 containing the nanowires 406, and the excess amount is discharged from the outlet 454.

[0128] like Fig. 9As shown in FIG. 4B , the aqueous solution 471 is discharged from the outlet 454 to the outside of the flow path space 452. In some embodiments, a water-absorbing material (not shown) may be brought into contact with the aqueous solution 471 at the outlet 454 to absorb the aqueous solution 471. As the aqueous solution 471 is discharged from the outlet 454, air 472 enters the flow path space 452 from the inlet 453. As the air 472 is introduced, the aqueous solution 471 disappears from the hydrophobic surface 402, and the aqueous solution 471 remains in the dot nanowire space 480 confined by the nanowire 406 to form droplets.

[0129] like Fig. 9 As shown in FIG. 3C , when sufficient or excess aqueous solution 471 is discharged from flow path space 452, aqueous solution 471 is isolated only in point nanowire space 480 defined by nanowires 406 in flow path space 452. Flow path space 452 other than point nanowire space 480 is occupied by air 472.

[0130] Fig. 9 In the embodiments shown in A to 9C, on the surface of the substrate 401, the hydrophobic region 402 and a plurality of hydrophilic dot nanowire spaces 480 surrounded thereby are used to isolate the aqueous solution 471 within the dot nanowire spaces 480, thereby forming so-called microdroplets.

[0131] <Other examples of isolation>

[0132] In some embodiments, a gas can be introduced to the substrate surface, and the flow, pressure, momentum, etc. of the gas can be used to remove the aqueous solution from the hydrophobic surface. In this case, the aqueous solution located in the hydrophobic area is easily removed. On the other hand, the aqueous solution located in the hydrophilic nanowire area remains in its position. Alternatively, the gas can be introduced to the substrate surface under conditions that allow the aqueous solution to remain in the dot nanowire space and remove the aqueous solution from the hydrophobic surface. As a result, the aqueous solution can be isolated in the dot nanowire space.

[0133] In some embodiments, a non-aqueous solution (also referred to as non-aqueous solution or non-aqueous liquid in this specification) may be further introduced into the flow path space 452. Fig. 9 As shown in FIG. 4D , a non-aqueous solution 473 is introduced from the inlet 453 into the flow path space 452. The non-aqueous solution 473 fills the flow path space 452 toward the outlet 454, and finally fills all the spaces (except the nanowire space 480) in the flow path space 452. The excess amount can be discharged from the outlet 454. In this state, substantially all the nanodot spaces 480 located in the flow path space 452 retain the aqueous solution 471 and are enclosed in the non-aqueous solution 473.

[0134] Fig. 9In the embodiment shown in D, the isolated aqueous solution 471 is enclosed in the dot nanowire space 480 by the non-aqueous solution 473 surrounding it. This can prevent, for example, evaporation of the aqueous solution 471, and enables stable measurement or observation.

[0135] <Introduction of non-aqueous solution>

[0136] Fig. 9 In some embodiments, the aqueous solution 471 outside the dot nanowire space 480 is replaced with air. Fig. 9 The replacement step with air shown in B to 9C can be changed to replacement with a non-aqueous solution. The non-aqueous solution can be a kind of oil. At this time, the aqueous solution located in the hydrophobic area is replaced by the introduced non-aqueous solution. On the other hand, it is difficult for the non-aqueous solution to enter the hydrophilic dot nanowire space 480. Therefore, its aqueous solution is difficult to be replaced by the non-aqueous solution. As a result, the aqueous solution is isolated from the nanowire area and remains. Furthermore, the dot nanowire space that retains the aqueous solution can be closed with a non-aqueous solution.

[0137] <Isolation of objects in droplets>

[0138] In some embodiments, an aqueous solution containing an object (object molecule, etc.) can be introduced onto the substrate surface, and the aqueous solution can be removed from the hydrophobic surface, thereby isolating the object within the dot nanowire space.

[0139] Reference Fig.10 A method for isolating an object within a dot nanowire space according to one embodiment is described below.

[0140] like Fig.10 As shown in FIG. 1A, a flow path device 500 is provided. The flow path device 500 has a flow path space 552 formed inside by bonding a first substrate 501 and a second substrate 551 to each other. The first substrate 501 has a hydrophobic film 502 on its entire surface. A plurality of nanowire regions 505A are arranged thereon, where a plurality of nanowires 506 are arranged. These are arranged inside the flow path space 552. The second substrate 551 has an inlet 553 and an outlet 554 for a liquid.

[0141] The aqueous solution 571 containing the target substance 575 is introduced into the flow path space 552 from the inlet 553 and fills the flow path space 552 containing the nanowires 506, and the excess amount is discharged from the outlet 554 ( Fig.10 B).

[0142] Using the method of any embodiment of the present invention or other methods, the aqueous solution 571 is placed in the dot nanowire space 580 or isolated as the dot nanowire space 580. At this time, the object substance 575 can be isolated in the dot nanowire space 580 ( Fig.10 C).

[0143] In some embodiments, a non-aqueous solution may be further introduced into the flow path space 552. Fig.10 As shown in FIG. 5D , a non-aqueous solution 573 is introduced from the inlet 553 into the flow path space 552. The non-aqueous solution 573 fills the flow path space 552 toward the outlet 554, and finally fills all the spaces (except the nanowire spaces 580) in the flow path space 552. In this state, substantially all the nanodot spaces 580 located in the flow path space 552 retain the aqueous solution 571 and the target substance 575, and are enclosed in the non-aqueous solution 573.

[0144] In some embodiments, the nanowires can be made to capture objects. Fig.10 In C and 10D, the target substance 575 is captured on the surface of the nanowire 506 in the nanowire space 580. In some embodiments, the nanowires may not capture the target but be confined in the nanowire space.

[0145] Furthermore, in Fig.10 In the embodiment shown in D, the isolated aqueous solution 571 and the target substance 575 contained therein are enclosed in the dot nanowire space 580 by the non-aqueous solution 572 surrounding them. This can prevent, for example, evaporation of the aqueous solution 571, and enables stable measurement or observation.

[0146] <Example 1 of isolating the target substance>

[0147] In this example, extracellular vesicles (EVs) were captured in the nanowire space. Specifically, EVs (2.3×10 9 The aqueous solution was introduced into the Figure 8 The same model device is shown.

[0148] In this embodiment, the aqueous solution is introduced into the device and incubated for 2 hours to allow mCherry to bind to EV. It is believed that during this period, the capture of EV by the nanowires is also promoted. Then, the aqueous solution in the flow path is sucked from the outlet with an absorbent sheet, the device flow path is rinsed with PBS to form droplets, and oil is introduced into the flow path to protect the droplets.

[0149] Fig.11 The figure shows a fluorescence microscope image (A) and a conventional optical microscope image (B) observed from above. The position of the dot nanowire space is confirmed in the optical microscope image (B). According to the fluorescence microscope image (A), the fluorescence of mCherry is confirmed to be confined to the position of its dot nanowire space. It can be understood that EV is isolated and captured in the dot nanowire space.

[0150] Other methods of isolating the target substance will be described below.

[0151] <Example 2 of isolating the target substance>

[0152] The preparation of the aqueous solution was the same as in Example 1. In this example, after the aqueous solution was introduced into the device, the aqueous solution in the flow path was first absorbed from the outlet using a water-absorbing sheet, and then incubated for 2 hours. Then, the device flow path was rinsed with PBS to form droplets, and oil was introduced into the flow path to protect the droplets.

[0153] Similar to Example 1, based on the fluorescence microscope image, it can be understood that EVs are isolated and captured in the dot nanowire space.

[0154] <Example 3 of isolating the target substance>

[0155] The preparation of the aqueous solution is the same as in Example 1. In this example, after the aqueous solution is introduced into the device, it is kept in a vacuum dryer for 2 hours. Thus, the water in the aqueous solution evaporates, and the remaining aqueous solution is isolated in the dot nanowire space. Then, the device flow path is rinsed with PBS to form droplets, and oil is introduced into the flow path to protect the droplets.

[0156] Similar to Example 1, based on the fluorescence microscope image, it can be understood that EVs are isolated and captured in the dot nanowire space.

[0157] Fig.12 : shows the frequency (vertical axis) relative to the fluorescence intensity (horizontal axis) at the position corresponding to the dot nanowire space in the fluorescence microscope images of Examples 1 to 3 above.

[0158] about Fig.12 The frequency distribution in A (Example 1) is Fig.12 B (Example 2) The high fluorescence intensity area becomes wider. This indicates that there are multiple fluorescent markers in one point. In other words, the concentration of fluorescent markers in each nanowire space is high. That is, the capture efficiency of EV as the target substance is high.

[0159] In Example 2, when the aqueous solution is sucked, the target substance and fluorescent marker contained therein are also discharged from the flow path. On the other hand, in Example 1, when incubated, the target substance and fluorescent marker are captured by the nanowires, which is considered to contribute to Fig.12 In A, the distribution at high fluorescence intensity becomes broad.

[0160] Fig.12 The frequency distribution in C (Example 3) is also Fig.12 B (Example 2) The high fluorescence intensity is broadened. Similarly, this indicates that there are multiple fluorescent markers in one point. In other words, the concentration of fluorescent markers in each nanowire space is high. That is, the capture efficiency of EV as the target substance is high.

[0161] In Example 3, the concentration of the target substance increases with the evaporation of water due to drying, and is highest in the dot nanowire space where the aqueous solution finally remains. As a result, it is believed that the target substance and fluorescent marker captured in the dot nanowire space at a high concentration contribute to the broadening of the distribution at high fluorescence intensity.

[0162] Fig.12 The distribution of C relative to Fig.12 As for the distribution of A, it is higher at high fluorescence intensity, but no obvious difference is observed. Fig.12 B (Example 2) can more efficiently isolate and capture the target substance within the dot nanowire space.

[0163] <Quantitative analysis using digital droplet array>

[0164] A digital droplet array (nano-dot space array) of one embodiment of the present invention can be used for quantitative analysis. The substrate, flow path device and method of the present invention can be used for digital analysis. In some embodiments, the aqueous solution to be introduced can contain the substance to be measured. The aqueous solution can be subjected to endpoint dilution or limiting dilution (either dilution is not strictly defined and distinguished, and it should be understood that they are not used mutually exclusive). By introducing and isolating the aqueous solution, the substance to be measured is enclosed in the nano-dot space.

[0165] In some embodiments, the substance to be measured may contain an optical or fluorescent marker (hereinafter sometimes referred to as an optical marker) and may be bound thereto. In some embodiments, the substance to be measured may be bound to an optical or fluorescent marker before being introduced into a fluid device. In some embodiments, the substance to be measured may be bound to an optical or fluorescent marker after being introduced into a fluid device and before or after the aqueous solution is isolated. For example, it can be said that the first aqueous solution contains the substance to be measured, the second aqueous solution contains the optical marker, and the first aqueous solution and the second aqueous solution are introduced into the fluid device in any order. Thus, the substance to be measured can be bound to an optical or fluorescent marker in a point space (before or after isolation).

[0166] In some embodiments, the assay object, optical marker, or a complex thereof can be captured by the nanowire. "Capture" refers to adsorption or binding to the nanowire by hydrogen bonding, van der Waals forces, ionic bonding, covalent bonding, etc., but the mechanism may not be limited. Nanowires are known to capture various biomolecules or biomaterials. Therefore, the point space with nanowires can easily and efficiently capture biomaterials and isolate them, making it easy to digitally quantify them.

[0167] <Digital droplet array measurement: Example 1 (digital immunoassay)>

[0168] Using the digital droplet array of one embodiment of the present invention, single-molecule digital immunoassay can be performed. One or zero molecules can be enclosed in each nanodot space to create a digital droplet array (nanodot space array) for identifying them.

[0169] As an example, the inventors confirmed using a series of multiple concentrations (not shown) that the concentration of the complex (conjugate) with the anti-CD9 antibody can be measured using biotin. 90 μL of each conjugate solution was introduced into the above-mentioned flow path device and left at room temperature for 1 hour. The device was then thoroughly cleaned.

[0170] Then, as a biotin label, a 10 μg / mL conjugate of streptavidin and β-galactosidase was prepared, 90 μL of the conjugate solution was introduced into the above-mentioned flow channel device, and allowed to stand at room temperature for 1 hour, and then the device was thoroughly washed.

[0171] Next, 10 μM Tokyo Green (registered trademark, the same applies hereinafter) was prepared as a fluorescent substrate for detecting β-galactosidase, and 90 μL of this solution was introduced into the above-mentioned flow channel device.

[0172] Then, 90 μL of oil (trade name FC40, 3M (registered trademark), Fluorinator (registered trademark)) was introduced into the device, and the solution after the reaction was isolated in the dot nanowire space and left overnight.

[0173] Fluorescence microscopy was then performed to confirm that digital immunoassay could be performed.

[0174] <Digital droplet array measurement: Example 2 (ddPCR)>

[0175] The digital droplet array of one embodiment of the present invention can be used to perform digital PCR (polymerase chain reaction), namely digital droplet PCR (ddPCR). PCR can be real-time PCR (qPCR).

[0176] First, mix the sample nucleic acid with primers and probes. Introduce the mixed solution into the nanodot space array device. Then, introduce oil into the device to isolate the mixed solution in the nanodot space. Perform PCR amplification in each nanodot space. Measure the fluorescence of each nanodot space. Determine the positivity / negativity of each nanodot space based on the measurement results. Count the number of nanodot spaces judged as positive and perform absolute quantification. Finally, the concentration of the sample nucleic acid (copy / μL) can be calculated.

[0177] <Digital droplet array measurement: Example 3 (digital ELISA)>

[0178] In the indirect method, a primary antibody is reacted with the target protein, and then a secondary antibody labeled with an enzyme against the primary antibody is reacted. For the complex, the mixed solution is introduced into a nanodot space array device. Then, a substrate is introduced into the device. Then the nanodot space is isolated. The absorbance of each nanodot space is measured. The positive / negative of each nanodot space is determined based on the measurement results. The number of nanodot spaces judged as positive is counted and absolutely quantified. Finally, the concentration of the target protein can be calculated. (Reference: Kim SH, et al., “Large-scale femtoliter droplet array for digital counting of single biomolecules”, Lab on a Chip Online Edition: 2012 / 8 / 15 (Japan time), doi: 10.1039 / C2LC40632B.)

[0179] The steps of mixing substances, solutions, etc. are not limited to the above. For example, a primary antibody may be reacted with an antigen, followed by a secondary antibody, and an enzyme may be introduced therein. Alternatively, other steps may be used.

[0180] An example of detecting target miRNA in EV is described below.

[0181] First, lipid nanoparticles are prepared. The lipid nanoparticles with positive surface charges contain a molecular beacon having a sequence complementary to the sequence of the target miRNA. Furthermore, NH2-PEG molecules are embedded in the membrane of the lipid nanoparticles.

[0182] The surface of the nanowire in the nanodot space is activated to bind the lipid nanoparticles. For example, the surface of the nanowire is activated with N-hydroxysuccinimide (NHS). NH2 can be covalently bonded thereto, and the lipid nanoparticles can be fixed to the nanowire via an amide bond. That is, a structure such as NW-CONH-lipid nanoparticles is prepared.

[0183] The target exosomes are introduced into the nanodot space to form droplets. Thus, the exosomes and lipid nanoparticles fuse in one nanodot space. As a result, the target miRNA in the exosome reacts with the molecular beacon in the lipid nanoparticle. When the target miRNA exists inside the exosome, the complementary nucleic acid sequence of the molecular beacon binds to the nucleic acid sequence, and the fluorescent pigment of the molecular beacon leaves the quencher and emits light. The presence or absence of luminescence in each nanodot space can be observed in a large number of nanodot spaces.

[0184] The digital droplet array of the present invention and its use are not limited to the above, and can also be used for other determinations. For example, protein binding analysis can be performed.

[0185] In some embodiments, the nanodot space of the present invention can be used as a detection field for fluorescently labeled antigens. Various processes are possible. Non-limiting examples include the following:

[0186] 1) First, introduce the primary antibody into the nanodot space, then introduce the antigen, and then introduce the fluorescently labeled secondary antibody.

[0187] 2) First, introduce the primary antibody into the nanodot space, then introduce the antigen, then introduce the secondary antibody, and then introduce the fluorescently labeled IgG.

[0188] 3) First, introduce the primary antibody into the nanodot space, then introduce the antigen. Then introduce the secondary antibody. Introduce the fluorescent substrate into it and observe the enzyme reaction.

[0189] 4) First, the antigen is introduced into the nanodot space, followed by the fluorescently labeled primary antibody.

[0190] In 1) to 3), the antigen can be introduced into the primary antibody.

[0191] In some embodiments, a reaction solution containing a target molecule, a polymerase, and a primer can be introduced into the nanodot space of the present invention, thereby being used as a detection field for their reaction. Various processes are possible. Non-limiting examples include the following:

[0192] 1) First, the target molecule is fixed to the nanowire within the nanodot space, and then a reaction solution containing polymerase and primers is introduced.

[0193] 2) First, the polymerase is fixed to the nanowire in the nanodot space, and then the reaction solution containing the target molecule and primers is introduced.

[0194] 3) First, the primer is fixed to the nanowire in the nanodot space, and then a reaction solution containing the target molecule and polymerase is introduced.

[0195] 4) First, the target molecule, polymerase and primer are fixed to the nanowire in the nanodot space, and then the reaction solution is introduced. In this case, the reaction can be controlled by the salt concentration.

[0196] <Further Embodiment>

[0197] Reference Fig.13 Some further implementations are described. Fig.13 After the relevant description, in the section "Experimental Methods", specific examples of the production of nanowire devices, EV capture and fluorescence measurement are described.

[0198] Provide a device having a spatial array of nanodots ( Fig.13 A1). A plurality of nanodot nanowires are arranged in an array in the flow path of the device ( Fig.13 A1c)(also called dot array nanowire)( Fig.13 A1b). Then, the target substance ( Fig.13 EV in the middle) is introduced into the flow path ( Fig.13 A2). Then the labeled antibody is introduced into the flow channel to bind to the target substance ( Fig.13 A3). Start the luciferase reaction ( Fig.13 A4), and perform fluorescence measurement.

[0199] like Fig.13 As shown in B2 to 13B4, as one embodiment, EVs that have been labeled with a fluorescent marker can be used as the target substance. Fig.13 In B, as a non-limiting example, EV1301b derived from HEK293T cells, expressing CD9 protein 1303 and labeled with mCherry1302 (hereinafter referred to as "HEK293T-CD9-mCherry cell-derived EV") was used.

[0200] EV1301b from HEK293T-CD9-mCherry cells introduced into the flow channel is adsorbed on nanowire 1306 ( Fig.13 B2). Then, the anti-CD9-biotin complex 1311 as a labeled antibody is introduced into the flow channel to recognize the CD9 protein 1303 on the surface of EV1301b to form an antigen-antibody complex ( Fig.13 B3). In which, avidin complex 1321 ( Fig.13 The streptavidin-β-galactosidase complex in the middle) binds to the biotin in the biotin complex 1311 through the avidin-biotin interaction. Then the fluorescent substrate ( Fig.13 The fluorescence intensity of the nanodots was measured by using 1331 and 1341 from Tokyo Green (Gurion Chemical Co., Ltd.). Fig.13 B4) In the optical measurement, both mCherry and TokyoGreen were measured.

[0201] The fluorescence intensity actually obtained based on the above scheme is shown in Fig.14 It was clearly confirmed that the fluorescence intensity of mCherry ( Fig.14 a) and TG( Fig.14 b) was higher than the negative control without EV in any case. In addition, the mCherry point and the TG point were observed at approximately the same point using a fluorescence microscope. Thus, it was confirmed that the target EV was observed by TG.

[0202] like Fig.13 As shown in C2 to 13C4, as one embodiment, a target substance that is not labeled with a fluorescent marker or the like (unlabeled) can be used. Fig.13 In C, as a non-limiting example, EV1301c derived from HEK293T cells and expressing CD9 protein 1303 (hereinafter referred to as "EV derived from HEK293T-CD9 cells") was used.

[0203] EV1301c from HEK293T-CD9 cells introduced into the flow channel is adsorbed on the nanowire 1306 ( Fig.13 B2). Then, the anti-CD9-biotin complex 1311 as a labeled antibody is introduced into the flow channel to recognize the CD9 protein 1303 on the surface of EV1301c to form an antigen-antibody complex ( Fig.13 C3). In this process, the avidin complex 1321 is bound to the biotin of the biotin complex 1311 through the avidin-biotin interaction. Then, the fluorescent substrates 1331 and 1341 are introduced to measure the fluorescence intensity of the nanodots ( Fig.13 C4). In the optical measurement, Tokyo Green was measured.

[0204] like Fig.13 As shown in D2 to 13D4, as an embodiment, an antibody that recognizes a certain protein of a target substance can be arranged on the surface of the nanowire 1306. As a non-limiting example, an anti-CD9 antibody 1351 can be fixed on the surface of the nanowire 1306 ( Fig.13 D2).

[0205] The EV1301d from HEK293T-CD9 cells introduced into the flow channel approaches the surface of the nanowire 1306, and the CD9 on its surface and the anti-CD9 antibody 1351 form an antigen-antibody complex ( Fig.13 C2). Then, the anti-CD9-biotin complex 1311 as a labeled antibody is introduced into the flow channel to recognize the CD9 protein 1303 on the surface of EV1301c to form an antigen-antibody complex ( Fig.13 C3). In this process, the avidin complex 1321 is bound to the biotin of the biotin complex 1311 through the avidin-biotin interaction. Then, the fluorescent substrates 1331 and 1341 are introduced to measure the fluorescence intensity of the nanodots ( Fig.13 C4). In the optical measurement, Tokyo Green was measured.

[0206] <Example>

[0207] As Fig.13In one example of the embodiment shown in D2 to 13D4, an attempt was made to detect high-grade serous ovarian cancer (HGSOC), which is a subtype of epithelial ovarian cancer (EOC). As membrane proteins expressed on the surface of EVs derived from HGSOC cells, two proteins, tight junction protein-3 (CLDN3) and tumor-associated calcium signal transducer 2 (TACSTD2), which are believed to be highly expressed in EOC tissues, were selected.

[0208] The surface of the nanowires was modified with anti-CD9 antibodies. Urine samples were collected from HGSOC patients and non-cancer patients. Each urine sample (N=5) was appropriately adjusted and introduced into the device. Then, fluorescently labeled CLDN3 antibody and fluorescently labeled TACSTD2 antibody were introduced into the device respectively. Other or detailed experimental conditions are shown below.

[0209] Fig.15 The results are shown in . The maximum value (maximum value of Gaussian distribution) of the fluorescence intensity distribution of "no EV" (no EV) as a negative control is set as a threshold, and the frequency of intensities greater than the threshold is plotted. It is clearly shown that HGSOC cells are detected using CD9 and CLDN3 as target proteins. In all samples, the use of CD9 and TACSTD2 as target proteins for detecting HGSOC cells was not shown, but as a tendency, this possibility was shown.

[0210] In this embodiment, EV is used as the target substance, anti-CD9 antibody is fixed to the surface of the nanowire, and fluorescently labeled CLDN3 antibody or fluorescently labeled TACSTD2 antibody is used, but the embodiments of the present invention are not limited to this. In this embodiment, EV is used as the target substance, but it can also be other biological molecules or other target substances. In this embodiment, these two proteins are detected, but the present invention is not limited to these. Other membrane proteins can also be selected as the detection object. The membrane protein of the antibody fixed to the nanowire and the target biological molecule to be captured by the nanowire can be appropriately selected. Similarly, the labeled antibody and the membrane protein of the target biological molecule recognized by it can be appropriately selected. Generally, a variety of antibodies that recognize the target substance can be used. The first antibody can be fixed to the surface of the nanowire so that the introduced target substance is bound to it. The second antibody can be fluorescently labeled so that it is bound to the target substance. The target substance can be bound to the first antibody and fixed to the surface of the nanowire to form a complex bound to the fluorescently labeled second antibody. In this state, a fluorescent reaction can be performed and its fluorescence can be detected. The primary and secondary antibodies of the fluorescent substrate and the fluorescent marker can be appropriately selected.

[0211] Fig.13 B2 and Fig.13The capture of EV in C2 is believed to be based on the charge reaction between EV1301b and nanowire 1306. Fig.13 The capture of EV in D2 is believed to be based on the immunoaffinity between the anti-CD9 antibody 1351 immobilized on the nanowire 1306 and the CD9 protein 1309 expressed on the surface of EV1301b. However, these capture mechanisms should not be interpreted restrictively, and other scientifically possible mechanisms are not prevented from being proposed.

[0212] <Experimental methods>

[0213] <Fabrication of dot nanowire devices>

[0214] In the above-mentioned embodiment, a CYTOP film was first formed on a synthetic quartz substrate by spin coating, and then a ZnO seed region was formed by patterning. The nanowires were grown on the ZnO seed by a hydrothermal method using 30 mM zinc nitrate hexahydrate (Zn(NO3)2·6H2O, Thermo Fisher Scientific) and hexamethylenetetramine (HMTA, C6H 12 N4, Wako Pure Chemical Industries, Ltd.) was synthesized at 95°C for 1 hour. The flow path chamber was produced by installing a flow path block of a cycloolefin polymer on the synthesized nanowire substrate.

[0215] <Immobilization of anti-CD9 antibody to nanowires>

[0216] The flow channel of the device was filled with 1 mg / mL of anti-human CD9 antibody (Cosmobio), left to stand for 1 hour, and then washed with PBS. Thus, the surface of the nanowires was modified with the anti-human CD9 antibody.

[0217] <Preparation of urine samples>

[0218] After collecting urine samples from HGSOC patients and non-cancer subjects, the urine samples were centrifuged (10 minutes, 4°C, 300×g, then 10 minutes, 4°C, 3000×g) to remove apoptotic bodies and filtered with a 0.22 μm filter (Merck Millipore Ltd.). Then, the filtered urine samples were incubated in the well plate analysis system according to the procedure.

[0219] <EV Capture>

[0220] 6 μL of the above-prepared urine sample was introduced into the device. Then, a blocking solution (KPL 10% BSA blocking solution, SeraCare) was introduced and incubated at 37°C for 1 hour. Then, PBS was added to wash the remaining sample.

[0221] <Identification using labeled antibodies>

[0222] As the primary antibody, a biotinylated anti-human CLDN3 antibody (Origi Tech) or a biotinylated anti-human TACSTD2 antibody (Exbio Praha as) was used and introduced into the device at 100 nM. After incubation at room temperature for 1 hour, wash 3 times with PBS. Then, streptavidin-β gal (SIGMA) was used as an enzyme marker and introduced into the device at 1 nM. After incubation at room temperature for 1 hour, wash 3 times with PBS. In order to allow the enzyme reaction in the dot array nanowires to proceed, the channel was filled with 10 μM of the substrate TokyoGreen (TG) (Wuling Chemical Co., Ltd.), and the TG solution in the channel was replaced with FC-40 (3M Japan Co., Ltd.) and incubated at 37 ° C for 20 hours.

[0223] <Fluorescence observation>

[0224] For fluorescence imaging, observation was performed using a confocal microscope (Leica Microsystems). Images were taken at 1024×1024 pixels and 16 bits. The fluorescence image of mCherry was obtained using excitation at 561 nm and 600 to 800 nm, and the fluorescence image of TG was obtained using excitation and luminescence at 488 nm and 500 to 550 nm. The fluorescence intensity of dot array nanowires (dot nanowires) was analyzed using AquaCOSMOS software (Hamamatsu Photonics Co., Ltd.).

[0225] The present invention also provides the following embodiments:

[0226] A001

[0227] A method for treating an aqueous solution and / or a method for forming microdroplets, comprising the following steps:

[0228] Providing a substrate / device / channel having a surface, wherein the surface is a surface having a hydrophobic surface (region) and a region (nanowire region) surrounded by the hydrophobic surface, wherein the nanowire region has hydrophilic nanowires;

[0229] introducing an aqueous solution onto the surface of the substrate; and

[0230] The aqueous solution is isolated from the nanowire region.

[0231] A002

[0232] A method according to A001 or any one of the embodiments, wherein the nanowire regions each comprise a plurality of nanowire regions surrounded by the hydrophobic surface.

[0233] A011

[0234] The method according to A001, A002 or any one of the embodiments, wherein the step of isolating the aqueous solution from the hydrophilic nanowires comprises the step of removing the introduced aqueous solution from the hydrophobic surface.

[0235] A012a

[0236] The method according to A011 or any one of the embodiments, wherein the step of isolating the aqueous solution from the hydrophilic nanowires comprises the step of allowing a water-absorbing material to absorb the aqueous solution located on the hydrophobic surface.

[0237] A012b

[0238] The method according to A011 or any one of the embodiments, wherein the step of isolating the aqueous solution from the hydrophilic nanowires comprises the step of introducing a gas (eg, air) onto the surface of the substrate.

[0239] A012c

[0240] According to the method described in A011, A012a, A012b or any one of the embodiments, the step of isolating the aqueous solution from the hydrophilic nanowires includes the step of introducing a non-aqueous solution onto the surface of the substrate.

[0241] A020

[0242] A method according to any one of A001 to A012c or any one of the embodiments, wherein the method for treating an aqueous solution and / or the method for forming microdroplets is a method for performing digital analysis.

[0243] A021

[0244] The method according to A020 ​​or any embodiment, wherein the above-mentioned digital analysis is selected from the group consisting of ddPCR (qPCR), digital ELISA and protein binding analysis.

[0245] A022

[0246] The method according to any one of A001 to A021 or any one of the embodiments, wherein the step of introducing the aqueous solution onto the surface of the substrate includes the step of introducing a (first) aqueous solution containing the target substance.

[0247] A023

[0248] The method according to A022 or any one of the embodiments, wherein the aqueous solution containing the target substance is subjected to limiting dilution or endpoint dilution.

[0249] A025

[0250] A method according to A022 or A023, or any one of the embodiments, further comprising the following steps:

[0251] After isolating the aqueous solution from the nanowire region, the target substance present in the isolated nanowire region is measured or detected.

[0252] A031

[0253] The method according to any one of A022 to A025 or any one of the embodiments further comprises the step of causing the hydrophilic nanowire to capture the target substance.

[0254] A032

[0255] The method according to A031 or any one of the embodiments further comprises the step of detecting the target substance captured by the hydrophilic nanowires.

[0256] A041

[0257] A method according to any one of A022 to A032 or any one of the embodiments, wherein the aqueous solution comprises a first aqueous solution containing a target substance and a second aqueous solution containing a marker for identifying the target substance.

[0258] A042

[0259] According to the method described in any one of A022 to A032 or any one of the embodiments, the step of introducing an aqueous solution into the surface of the substrate comprises: introducing a first aqueous solution containing a target substance; and introducing a second aqueous solution containing a marker for identifying the target substance.

[0260] A043

[0261] The method according to A041 or A042, or any one of the embodiments, further comprises the step of causing the marker to recognize the target substance.

[0262] A044a

[0263] A method according to A041 or A042, or any embodiment, wherein the method further comprises the following steps:

[0264] introducing a first aqueous solution containing a target substance so that the nanowires capture the target substance; and

[0265] A second aqueous solution containing a marker that recognizes the target substance is introduced so that the marker recognizes the target substance captured by the nanowires.

[0266] A044b

[0267] A method according to A041 or A042, or any embodiment, wherein the method further comprises the following steps:

[0268] introducing a second aqueous solution containing a marker of a recognition target substance so that the nanowires capture the marker; and

[0269] A first aqueous solution containing a target substance is introduced so that the marker captured by the nanowires recognizes the target substance.

[0270] A044c

[0271] A method according to A041 or A042, or any embodiment, wherein the method further comprises the following steps:

[0272] introducing a first aqueous solution containing a target substance;

[0273] introducing a second aqueous solution containing a marker for identifying the target substance;

[0274] allowing the marker to recognize the target substance; and

[0275] The nanowire is caused to capture a complex of the marker and the target substance.

[0276] A051

[0277] A method according to any one of A041 to A044c or any one of the embodiments, wherein the marker comprises an optical marker (fluorescent marker or absorbance marker).

[0278] A052

[0279] The method described in A051 or any embodiment further comprises the step of detecting the optical marker using an optical method.

[0280] A101

[0281] A method for detecting biomolecules, comprising the following steps:

[0282] providing a device having a plurality of dot nanowire regions;

[0283] Introducing target biomolecules onto the nanowire surface of the plurality of dot nanowire regions and capturing them;

[0284] introducing an optically labeled first antibody that recognizes the first membrane protein of the target biomolecule into the device, so that the first antibody recognizes the target biomolecule;

[0285] The optical markers described above are optically measured.

[0286] A111

[0287] A method for detecting biomolecules, comprising the following steps:

[0288] providing a device having a plurality of dot nanowire regions;

[0289] disposing a second antibody that recognizes a second membrane protein of the target biomolecule on the surface of the nanowires in the plurality of dot nanowire regions;

[0290] Introducing target biomolecules into the plurality of dot nanowire regions, so that the second antibody captures the target biomolecules;

[0291] introducing an optically labeled first antibody that recognizes the first membrane protein of the target biomolecule into the device, so that the first antibody recognizes the target biomolecule;

[0292] The optical markers described above are optically measured.

[0293] A121

[0294] A method according to A111 or any embodiment, wherein the first membrane protein is different from the second membrane protein; and / or the first antibody is different from the second antibody.

[0295] The preferred embodiments of the present invention are shown and described in this specification, but such embodiments are provided only as examples, which are obvious to those skilled in the art. It is not intended to limit the present invention by the specific examples provided in the specification. Although the present invention is described with reference to the aforementioned description, the description and illustration of the embodiments of this specification are not meant to be interpreted in a limiting sense. A person skilled in the art will think of various deformations, changes and substitutions without departing from the present invention. In addition, it should be understood that all modes of the present invention are not limited to the specific descriptions, compositions or relative ratios recorded in this specification that depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention recorded in this specification can be used when implementing the present invention. Therefore, the present invention intends to also cover such substitutions, changes, deformations or equivalents. It is intended that the scope of the present invention is defined by the claims of the present application, and these methods and structures in the claims and their equivalents are thereby covered.

Claims

1. A method for forming microdroplets, comprising the following steps: A device having a substrate surface is provided, wherein the substrate surface is a surface having a hydrophobic surface (region) and a plurality of regions (nanowire regions) surrounded by the hydrophobic surface, and the nanowire regions have hydrophilic nanowires; introducing an aqueous solution onto the surface of the substrate; and The aqueous solution is isolated from the nanowire region.

2. The method according to claim 1, wherein: The step of isolating the aqueous solution from the hydrophilic nanowires includes the step of removing the introduced aqueous solution from the hydrophobic surface.

3. The method according to claim 1, wherein: The step of isolating the aqueous solution from the hydrophilic nanowires includes the step of allowing a water-absorbing material to absorb the aqueous solution located on the hydrophobic surface.

4. The method according to claim 1, wherein: The step of isolating the aqueous solution from the hydrophilic nanowires includes the step of introducing a gas onto the surface of the substrate.

5. The method according to claim 1, wherein: The step of isolating the aqueous solution from the hydrophilic nanowires includes the step of introducing a non-aqueous solution onto the surface of the substrate.

6. The method according to any one of claims 1 to 5, wherein: Method for processing aqueous solution / method for forming microdroplets for digital analysis.

7. The method according to claim 6, wherein: The digital analysis is selected from the group consisting of ddPCR (qPCR), digital ELISA and protein binding analysis.

8. The method according to claim 1, wherein: The step of introducing the aqueous solution onto the substrate surface includes the step of introducing a (first) aqueous solution containing a target substance.

9. The method according to claim 8, wherein: The method further comprises isolating the aqueous solution from the nanowire region and then measuring or detecting the target substance present in the isolated nanowire region.

10. The method according to claim 8, wherein: The method further comprises a step of causing the hydrophilic nanowires to capture the target substance.

11. The method according to claim 10, wherein: The method further comprises a step of detecting the target substance captured by the nanowire.

12. The method according to claim 8, wherein: The aqueous solution includes a first aqueous solution containing a target substance and a second aqueous solution containing a marker for identifying the target substance.

13. The method according to claim 8, wherein: The step of introducing the aqueous solution onto the surface of the substrate comprises the following steps: introducing a first aqueous solution containing a target substance; and A second aqueous solution containing a marker for identifying the target substance is introduced.

14. The method according to claim 12, wherein: The following steps are also included: With respect to the target substance, the marker is allowed to recognize the target substance.

15. The method according to claim 12, wherein: (Also) have the following steps: introducing a first aqueous solution containing a target substance so that the nanowires capture the target substance; as well as A second aqueous solution containing a marker that recognizes the target substance is introduced so that the marker recognizes the target substance captured by the nanowires.

16. The method according to claim 12, wherein: (Also) have the following steps: introducing a second aqueous solution containing a marker of a recognition target substance so that the nanowire captures the marker; as well as A first aqueous solution containing a target substance is introduced so that the marker captured by the nanowires recognizes the target substance.

17. The method according to claim 12, wherein: (Also) the following steps: introducing a first aqueous solution containing a target substance; introducing a second aqueous solution containing a marker for identifying the target substance; allowing the marker to recognize the target substance; as well as The nanowire is caused to capture a complex of the marker and the target substance.

18. The method according to any one or any embodiment of claims 12 to 17, wherein: The markers include optical markers (fluorescent markers or light-absorbing markers).

19. The method according to claim 18, wherein: The method further comprises the step of detecting the optical marker using an optical method.

20. A method for detecting a biomolecule, comprising the following steps: providing a device having a plurality of dot nanowire regions; disposing a second antibody that recognizes a second membrane protein of the target biomolecule on the surface of the nanowires in the plurality of dot nanowire regions; Introducing target biomolecules into the plurality of dot nanowire regions, so that the second antibody captures the target biomolecules; introducing an optically labeled first antibody that recognizes a first membrane protein of the target biomolecule into the device, so that the first antibody recognizes the target biomolecule; The optical marker is optically detected.

21. The method according to claim 20, wherein: The first membrane protein is different from the second membrane protein; and / or The first antibody is different from the second antibody.

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