Technique for arranging particles on substrate without agglutination

Abstract

To provide a novel technique for providing a substrate advantageous in producing an analytical sensor.SOLUTION: A substrate for producing an analytical sensor of the present disclosure has a specific feature (for example, monodispersion, a single layer, or specific density), thus can produce an analytical sensor capable of stable and highly accurate analysis. With the present disclosure, it is not necessary to remove excess after surplus fine particles are mounted on a substrate, so that reproducibility of density of the fine particles immobilized on the substrate is improved, and monodispersity and single-layer property are also improved.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a technique for arranging particles on a substrate used in a measurement technique without aggregation, and its applications. More specifically, the present disclosure relates to a substrate for fabricating a sensor for analyzing a detection target, on which a scaffold is arranged, a method for manufacturing the same, a sensor for analyzing a detection target, a method for manufacturing the same, and a method for analyzing the detection target. [Background technology]

[0002] Small extracellular vesicles (sEVs), such as exosomes, are a type of endoplasmic reticulum released from cells. They are lipid bilayer vesicles with a diameter of 20–200 nm. SEVs contain proteins and nucleic acids such as miRNA and mRNA within their interiors, and also possess proteins on their surface. Because SEVs are characterized by these substances, analyzing their characteristics is thought to enable us to infer the type of cell that secreted them. Furthermore, SEVs have been confirmed to exist in various body fluids and can be collected relatively easily.

[0003] Small extracellular vesicles secreted by cancer cells contain tumor-derived substances. Therefore, it is expected that analyzing the substances contained in small extracellular vesicles in body fluids will enable cancer diagnosis. Furthermore, because small extracellular vesicles are actively secreted by cells, it is expected that they will exhibit some characteristics even in the early stages of cancer. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides a novel technology for providing an advantageous substrate for fabricating analytical sensors. More specifically, the substrate for fabricating analytical sensors provided by the present disclosure has specific characteristics (e.g., monodispersity, monolayer, or specific density), allowing the fabrication of analytical sensors that enable stable and highly accurate analysis. The present disclosure eliminates the need to place an excess of microparticles on a substrate and then remove the excess, improving the reproducibility of the density of microparticles immobilized on a substrate and also improving the monodispersity and monolayer properties. In one preferred embodiment, the present disclosure provides a method for immobilizing particles by utilizing electrostatic interactions between the substrate and the particles, and arranging the particles on the substrate in a monodisperse and / or monolayer form, thereby enabling the reproducibility of a substrate on which monodisperse microparticles are immobilized and / or a substrate on which monolayer microparticles are immobilized.

[0005] The present disclosure provides, for example, the following:

[0006] (Item A1) A) a substrate body; B) A substrate for producing an analytical sensor, comprising particles monodispersely arranged on the substrate body. (Item A2-1) A) a substrate body; B) Particles, 1 x 10 3 pieces / mm 2 ~1×10 8 pieces / mm 2 and particles arranged on the substrate body at a density of 1000 nm to 1000 nm. (Item A2-2) A) a substrate body; B) Particles, 1 x 10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 and particles arranged on the substrate body at a density of 1000 nm to 1000 nm. (Item A2-3) 2. The analytical sensor fabrication substrate according to claim 1, wherein the particles contain a substance used in analytical sensors. (Item A3) The substrate for preparing an analytical sensor according to any one of the preceding items, wherein the particles include particles having a modifier integrated thereto. (Item A4) The substrate for producing an analytical sensor described in any one of the preceding items, wherein the substrate further includes C) a polymer matrix disposed on the substrate body, the polymer matrix having a recess that at least partially fits a target, and the particles are disposed in the recess. (Item B1A) A) a substrate body; B) a polymer matrix disposed on the substrate body, the polymer matrix having a recess that at least partially conforms to an object, and the particles disposed in the recess; C) a group for binding a signal substance arranged on the particle; D) a binding group for a specific binding molecule that binds to a molecule to be detected, the specific binding molecule being disposed on the particle, wherein the particle portion comprises 1×10 3 pieces / mm 2 ~1×10 8 pieces / mm 2 The convex analytical sensors are arranged on the substrate body at a density of 1000 nm to 1000 nm. (Item B1B) A) a substrate body; B) a polymer matrix disposed on the substrate body, the polymer matrix having a recess that at least partially conforms to an object, and the particles disposed in the recess; C) a group for binding a signal substance arranged on the particle; D) a binding group for a specific binding molecule that binds to a molecule to be detected, the specific binding molecule being disposed on the particle, wherein the particle portion comprises 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 The convex analytical sensors are arranged on the substrate body at a density of 1000 nm to 1000 nm. (Item B1C) A) a substrate body; B) a polymer matrix disposed on the substrate body, the polymer matrix having recesses that at least partially accommodate target molecules; C) a signal substance binding group arranged in the recess; D) a binding group for a specific binding molecule that binds to a molecule to be detected, disposed in the recess, wherein the recess contains 1×10 3 pieces / mm 2 ~1×10 8 pieces / mm 2 The analytical sensors are disposed on the substrate body at a density of 1000 nm to 1000 nm. (Item B1D) A) a substrate body; B) a polymer matrix disposed on the substrate body, the polymer matrix having recesses that at least partially accommodate target molecules; C) a signal substance binding group arranged in the recess; D) a binding group for a specific binding molecule that binds to a molecule to be detected, disposed in the recess, wherein the recess contains 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 The analytical sensors are disposed on the substrate body at a density of 1000 nm to 1000 nm. (Item B2) The recess is 1×10 4 pieces / mm 2 ~1×10 6 pieces / mm 2 The analytical sensor according to any one of the preceding items, wherein the sensor is present at a density of (Item C1A) A method for manufacturing a substrate for producing a convex analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate such that the particles are monodisperse and disposed within the substrate body; C) providing a polymer matrix material to the substrate having the particles fixed thereto; D) forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to conditions under which the polymer matrix polymerizes. encompassing, method. (Item C1) 1. A method for producing a substrate for preparing an analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate such that the particles are monodispersely disposed on the substrate body; C) providing a polymer matrix material to the substrate having the particles fixed thereto; D) forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to conditions under which the polymer matrix polymerizes; E) subjecting the substrate to conditions that dissociate the particles from the substrate, thereby forming recesses. encompassing, method. (Item C1-1) The step of adding particles so as to be arranged monodispersely may be carried out by adding particles to the substrate at a density of 1.0×10 per square mm of the substrate. 0 cells / µL to 2.5 x 10 8 at a concentration of 1.0 x 10 cells / µL 0 From 2.5 x 10 8 3. The method of claim 1, further comprising adding an amount of (Item C1-2) The step of adding particles so as to be arranged monodispersely may be carried out by adding particles to the substrate at a density of 1.0×10 per square mm of the substrate. 0 cells / µL to 1.0 x 10 10 at a concentration of 1.0 x 10 cells / µL 0 from 1.0 x 10 10 3. The method of claim 1, further comprising adding an amount of (Item C2A) 1. A method for manufacturing a convex analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate so that the particles are monodispersely disposed on the substrate body; and C) providing a raw material for a polymer matrix to the substrate on which the particles are fixed. D) forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to conditions under which the polymer matrix polymerizes; F) binding a substance required for analysis to the particles. encompassing, method. (Item C2) 1. A method for manufacturing an analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate such that the particles are monodisperse and disposed within the substrate body; C) providing a polymer matrix material to the substrate having the particles fixed thereto; D) forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to conditions under which the polymer matrix polymerizes; E) forming recesses by subjecting the substrate to conditions that dissociate the particles from the substrate; F) binding a substance required for analysis to the recess. encompassing, method. (Item C3A-1) 2.0×10 7 pieces / mm 2 a density of recesses formed in the analytical sensor, whereby 80% or more of the analytical sensors are produced with a coefficient of variation of 20% or less. (Item C3A-2) 1.0×10 10 pieces / mm 2 a density of recesses formed in the analytical sensor, whereby 80% or more of the analytical sensors are produced with a coefficient of variation of 20% or less. (Item C3B) A method for manufacturing the analytical sensor described in any one of the preceding items, wherein 80% or more of the analytical sensors are produced with a coefficient of variation of 20% or less by forming recesses on the substrate whose area occupancy is equal to or less than closest packing. (Item C4A) The step of disposing the particles in a monodisperse manner is carried out by disposing the particles in a monodisperse manner on a substrate. 3 pieces / mm 2 ~1×10 8 pieces / mm 2The method of any one of the preceding items, comprising a step of disposing the particles at a density of (Item C4B) The step of disposing the particles in a monodisperse manner is carried out by disposing the particles in a monodisperse manner on a substrate. 3 pieces / mm 2 ~1×10 10 pieces / mm 2 The method of any one of the preceding items, comprising a step of disposing the particles at a density of (Item C5A) In the step of forming the recesses, the monodispersed recesses are formed in a concentration of 1×10 3 pieces / mm 2 ~1×10 8 pieces / mm 2 The method of any one of the preceding items, wherein the granules are present at a density of (Item C5B) In the step of forming the recesses, the monodispersed recesses are formed in a concentration of 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 The method of any one of the preceding items, wherein the granules are present at a density of (Item C6) The method according to any one of the preceding items, wherein the step of disposing the particles includes a spin coating method, a method of dropping particles onto a substrate, a method of immersing a substrate in a particle dispersion, a method of pulling up a substrate from a particle dispersion, or a method of spraying a particle dispersion. (Item 1) A) a substrate body; B) A substrate for producing an analytical sensor, comprising particles arranged in a single layer on the substrate body. (Item 2) 2. The substrate for preparing an analytical sensor according to any one of the preceding items, wherein the particles are arranged without agglomeration. (Item 1A) A) a substrate body; B) Particles arranged on the substrate body without agglomerating. (Item 2A) 10. The substrate for preparing an analytical sensor according to any one of the preceding items, wherein the particles are in a single layer state. (Item 3) 10. The substrate for preparing an analytical sensor according to any one of the preceding items, wherein the particles include particles having a modifier integrated thereto. (Item 4) The substrate for preparing an analytical sensor according to any one of the preceding items, wherein the substrate further comprises C) a polymer matrix disposed on the substrate body, the polymer matrix having a recess that is adapted to at least a portion of the target, preferably in the sense of functioning as an analytical sensor, and the particles are disposed in the recess. (Item 5) A) a substrate body; B) a polymer matrix disposed on the substrate body, the polymer matrix having recesses that at least partially fit the target particles, and the particles are disposed in the recesses without agglomerating; C) a group for binding a signal substance arranged on the particle; D) a binding group for a specific binding molecule that binds to a molecule to be detected, disposed on the particle, wherein the particle has a density of 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 The convex analytical sensors are arranged on the substrate body at a density of 1000 nm to 1000 nm. (Item 6) A) a substrate body; B) a polymer matrix disposed on the substrate body, the polymer matrix having a recess that at least partially fits a detection target; C) a signal substance binding group arranged in the recess; D) a binding group for a specific binding molecule that binds to a molecule to be detected, disposed in the recess, wherein the recess contains 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2The analytical sensors are arranged on the substrate body at a density of 1000 nm to 1000 nm without clumping. (Item 7) The recess is 1×10 4 pieces / mm 2 ~1×10 6 pieces / mm 2 10. The analytical sensor of claim 9, wherein the analytical sensor is present without aggregation at a density of 0.1 to 1.25. (Item 8) A method for manufacturing a substrate for producing a convex analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate so that the particles are distributed throughout the substrate without agglomeration; C) providing a polymer matrix material to the substrate having the particles fixed thereto; D) forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to conditions under which the polymer matrix polymerizes. encompassing, method. (Item 9) 1. A method for producing a substrate for preparing an analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate so that the particles are disposed within the substrate body without agglomeration; C) providing a polymer matrix material to the substrate having the particles fixed thereto; D) forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to conditions under which the polymer matrix polymerizes; E) subjecting the substrate to conditions that dissociate the particles from the substrate, thereby forming recesses. encompassing, method. (Item 10) The step of adding particles so as to be arranged without agglomeration may involve adding particles to the substrate in an amount of 1.0×10 0 cells / µL to 1.0 x 10 10 at a concentration of 1.0 x 10 cells / µL, or per surface area of ​​substrate 0 pieces / mm 2 to 1.0×10 10 pieces / mm 28. The method of any one of the preceding items, comprising adding an amount of (Item 11) 1. A method for manufacturing a convex analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate so that the particles are distributed throughout the substrate without agglomeration; C) providing a polymer matrix material to the substrate having the particles fixed thereto; D) forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to conditions under which the polymer matrix polymerizes; E) binding a substance required for analysis to the particles. encompassing, method. (Item 12) 1. A method for manufacturing an analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate so that the particles are distributed throughout the substrate without agglomeration; C) providing a polymer matrix material to the substrate having the particles fixed thereto; D) forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to conditions under which the polymer matrix polymerizes; E) forming recesses by subjecting the substrate to conditions that dissociate the particles from the substrate; F) binding a substance required for analysis to the recess. encompassing, method. (Item 13) 1.0×10 10 pieces / mm 2 A method for manufacturing an analytical sensor described in any one of the preceding items, wherein by forming recesses at a density below 1000 kJ / cm, analytical sensors having a coefficient of variation of relative fluorescence intensity change in target substance detection of 20% or less are produced in 80% or more of the cases. (Item 14) A method for manufacturing an analytical sensor according to any one of the preceding items, wherein 80% or more of the analytical sensors are produced having a coefficient of variation of 20% or less in relative fluorescence intensity change in target substance detection by forming recesses on a substrate whose area occupancy is equal to or less than closest packing. (Item 15) The step of disposing without aggregation may comprise disposing 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 4. The method of any one of the preceding items, comprising disposing the particles at a density of (Item 16) 10. The method of any one of the preceding items, wherein the particles are in a single layer. (Item 17) In the step of forming the recess, the recess is formed to have a thickness of 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 8. The method of any one of the preceding items, wherein the granules are present at a density of (Item 18) 10. The method according to any one of the preceding items, wherein the step of disposing the particles comprises a spin coating method, a method of dropping particles onto a substrate, a method of immersing a substrate in a particle dispersion, a method of pulling up a substrate from a particle dispersion, or a method of spraying a particle dispersion.

[0007] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0008] By utilizing the technology disclosed herein, in a technique for creating pores for target molecules using particles using molecular imprinting technology, monodisperse or monolayer pores can be formed on a substrate, thereby improving the reproducibility of pore formation. The method disclosed herein is positioned as an important fundamental technology for creating uniform sensor substrates. [Brief explanation of the drawings]

[0009] [Figure 1-1] Figure 1-1 shows the NMR chart of polymer E2. [Figure 1-2] Figure 1-2 shows the NMR chart of polymer E3. [Figure 1-3] Figure 1-3 shows the NMR chart of polymer E4. [Figure 1-4] FIG. 1-4 shows the results of DLS measurements of particles in which silica particles and each of polymers E2 to E4 are integrated. [Figure 1-5] FIG. 1-5 shows the results of Z-potential measurement of particles in which silica particles are integrated with each of polymers E2 to E4. [Figure 1-6] FIG. 1-6 shows the results of DLS measurement and Z-potential measurement of particles in which silica particles and polymer (E2-0) are integrated. [Figure 2-1] FIG. 2-1 shows an image of the particle-immobilized substrate observed with a fluorescence microscope. [Figure 2-2] Figure 2-2 shows the results of hybrid cell count analysis of particle-immobilized substrates under a fluorescence microscope. [Figure 3-1] FIG. 3-1 shows images of particle-immobilized substrates in which silica particles and each polymer E4 are integrated, observed with a fluorescence microscope. [Figure 3-2] FIG. 3-2 shows images of particle-immobilized substrates in which silica particles and each polymer E3 are integrated, observed with a fluorescence microscope. [Figure 4] Figure 4 shows an image of the particle-immobilized substrate observed under a fluorescence microscope (glass substrate). [Figure 5-1] Figure 5-1 shows an image of the particle-immobilized substrate observed with a fluorescence microscope (polystyrene particles). [Figure 5-2] Figure 5-2 shows an image of the particle-immobilized substrate observed with a fluorescence microscope (polystyrene particles). [Figure 5-3] Figure 5-3 shows a fluorescent microscope image of the particle-immobilized substrate (polystyrene particles). [Figure 5-4] Figure 5-4 shows the hybrid cell count analysis results of particle-immobilized substrates under a fluorescence microscope (polystyrene particles). [Figure 6-1] FIG. 6-1 shows an image of the particle-immobilized substrate of Example 6 observed with a fluorescent microscope. [Figure 6-2] FIG. 6-2 shows an image of Example 6 observed under a fluorescent microscope after polymerization of the polymer layer on the substrate. [Figure 6-3] FIG. 6-3 shows an image of the substrate of Example 6 observed under a fluorescent microscope after the silica nanoparticles were removed. [Figure 6-4] Figure 6-4 shows the results of hybrid cell count analysis using a fluorescence microscope of particle-immobilized substrate. [Figure 7] FIG. 7 shows a fluorescence microscope image of a substrate on which a complex of His-Tag polymer and silica nanoparticles was immobilized. [Figure 8] Figure 8 shows a fluorescence microscope image and an SEM image of a substrate onto which a complex of cationic silica nanoparticles and anionic polymer Ex8 was immobilized. [Figure 9] FIG. 9 shows a fluorescence microscope image and an SEM image of a substrate on which particles were immobilized using biodegradable nanoparticles. [Figure 10-1] FIG. 10-1 shows a fluorescence microscope image and an SEM image of a substrate on which a complex of silica nanoparticles and a cationic polymer containing an aromatic component, E2P20, is immobilized. [Figure 10-2] FIG. 10-2 shows a fluorescence microscope image of a substrate on which a complex of polystyrene nanoparticles and a cationic polymer containing an aromatic moiety, E2P20, is immobilized. [Figure 11] FIG. 11 shows a fluorescence microscope image and an SEM image of a substrate on which silica nanoparticles to which His-tag and thiol groups have been introduced are immobilized. [Figure 12]FIG. 12 shows the change in fluorescence intensity on the substrate surface of a sensor fabricated without using particles. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present disclosure is described in further detail below.

[0011] Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will control.

[0012] (definition) First, we explain the terms and general techniques used in this disclosure.

[0013] As used herein, the term "substrate" refers to a substance that serves as the foundation of an analytical sensor. The substrate material may be, for example, a material selected from the group consisting of metals, metal oxides, glass, paper (cellulose), cloth, silicon dioxide, silicon, and resins, as well as combinations thereof. Metals include, but are not limited to, gold, silver, copper, aluminum, titanium, tungsten, and molybdenum. Resins include, but are not limited to, poly(meth)acrylate, polystyrene, ABS (acrylonitrile-butadiene-styrene copolymer), polycarbonate, polyester, polyethylene, polypropylene, nylon, polyurethane, silicone resin, fluororesin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, and vinyl chloride resin.

[0014] As used herein, "monodisperse" refers to a dispersed system in which the dispersed phase has a uniform size, and when referring to a substrate such as a substrate for fabricating an analytical sensor, it refers to particles being substantially uniformly arranged on the substrate. When particles are arranged in a monodisperse manner, they are arranged without agglomeration, which is advantageous. The density required to achieve monodispersity can vary depending on the intended target in the final analytical sensor, and is preferably about 10 3 pieces / mm 2 ~about 10 8 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 7 pieces / mm 2 , and more preferably about 10 4 pieces / mm 2 ~about 10 6 pieces / mm 2 The upper limit of the density is, for example, about 1 × 10 7 pieces / mm 2 , about 9×10 6 pieces / mm 2 , about 8×10 6 pieces / mm 2 , about 7×10 6 pieces / mm 2 , about 6×10 6 pieces / mm 2 , about 5×10 6 pieces / mm 2 , about 4×10 6 pieces / mm 2 , about 3×10 6 pieces / mm 2 , about 2×10 6 pieces / mm 2 , about 1×10 6 pieces / mm 2 The lower limit is, for example, about 1 × 10 3 pieces / mm 2 , about 2×10 3 pieces / mm 2 , about 3×10 3 pieces / mm 2 , about 4×10 3 pieces / mm 2 , about 5×10 3 pieces / mm 2, about 6×10 3 pieces / mm 2 , about 7×10 3 pieces / mm 2 , about 8×10 3 pieces / mm 2 , about 9×10 3 pieces / mm 2 , about 1 x about 10 4 pieces / mm 2 or preferably about 10 3 pieces / mm 2 ~about 10 10 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 9 pieces / mm 2 , and more preferably about 10 4 pieces / mm 2 ~about 10 8 pieces / mm 2 The upper limit of the density is, for example, about 1 × 10 10 pieces / mm 2 , about 9×10 9 pieces / mm 2 , about 8×10 9 pieces / mm 2 , about 7×10 9 pieces / mm 2 , about 6×10 9 pieces / mm 2 , about 5×10 9 pieces / mm 2 , about 4×10 9 pieces / mm 2 , about 3×10 9 pieces / mm 2 , about 2×10 9 pieces / mm 2 , about 1×10 9 pieces / mm 2 The lower limit is, for example, about 1 × 10 3 pieces / mm 2 , about 2×10 3 pieces / mm 2 , about 3×10 3 pieces / mm 2 , about 4×10 3 pieces / mm 2 , about 5×10 3 pieces / mm 2, about 6×10 3 pieces / mm 2 , about 7×10 3 pieces / mm 2 , about 8×10 3 pieces / mm 2 , about 9×10 3 pieces / mm 2 , about 1 x about 10 4 pieces / mm 2 Examples include:

[0015] As used herein, the term "monolayer" refers to a single layer of particles that do not overlap. When particles are arranged in a monolayer, they are arranged without aggregation, which is advantageous. The density required to achieve a monolayer can vary depending on the intended target in the final analytical sensor, and is preferably about 10 3 pieces / mm 2 ~about 10 8 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 7 pieces / mm 2 , and more preferably about 10 4 pieces / mm 2 ~about 10 6 pieces / mm 2 The upper limit of the density is, for example, about 1 × 10 7 pieces / mm 2 , about 9×10 6 pieces / mm 2 , about 8×10 6 pieces / mm 2 , about 7×10 6 pieces / mm 2 , about 6×10 6 pieces / mm 2 , about 5×10 6 pieces / mm 2 , about 4×10 6 pieces / mm 2 , about 3×10 6 pieces / mm 2 , about 2×10 6 pieces / mm 2 , about 1×10 6 pieces / mm 2 The lower limit is, for example, about 1 × 10 3pieces / mm 2 , about 2×10 3 pieces / mm 2 , about 3×10 3 pieces / mm 2 , about 4×10 3 pieces / mm 2 , about 5×10 3 pieces / mm 2 , about 6×10 3 pieces / mm 2 , about 7×10 3 pieces / mm 2 , about 8×10 3 pieces / mm 2 , about 9×10 3 pieces / mm 2 , about 1 x about 10 4 pieces / mm 2 or preferably about 10 3 pieces / mm 2 ~about 10 10 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 9 pieces / mm 2 , and more preferably about 10 4 pieces / mm 2 ~about 10 8 pieces / mm 2 The upper limit of the density is, for example, about 1 × 10 10 pieces / mm 2 , about 9×10 9 pieces / mm 2 , about 8×10 9 pieces / mm 2 , about 7×10 9 pieces / mm 2 , about 6×10 9 pieces / mm 2 , about 5×10 9 pieces / mm 2 , about 4×10 9 pieces / mm 2 , about 3×10 9 pieces / mm 2 , about 2×10 9 pieces / mm 2 , about 1×10 9 pieces / mm 2 The lower limit is, for example, about 1 × 10 3pieces / mm 2 , about 2×10 3 pieces / mm 2 , about 3×10 3 pieces / mm 2 , about 4×10 3 pieces / mm 2 , about 5×10 3 pieces / mm 2 , about 6×10 3 pieces / mm 2 , about 7×10 3 pieces / mm 2 , about 8×10 3 pieces / mm 2 , about 9×10 3 pieces / mm 2 , about 1 x about 10 4 pieces / mm 2 Examples include:

[0016] As used herein, "aggregation," when referring to particles, refers to the aggregation of multiple particles due to mutual attraction. "Absence of aggregation" or "no aggregation" refers to the presence of particles in a state where such aggregation is substantially absent (i.e., below the detection limit). Absence of aggregation typically refers to the absence of overlapping and fixed portions in any direction when measured by SEM, preferably 10% or less of the total, more preferably 5%, 4%, 3%, 2%, or 1% or less. The SEM measurement method is as follows: After vacuum drying the object to be measured, gold sputtering is performed, and conditions are set to achieve a magnification of 2000x or more. If a judgment cannot be made using SEM, observation can be made using an atomic force microscope. Using the technology disclosed herein, particles can be arranged on a substrate without aggregation.

[0017] As used herein, the term "single layer" typically refers to a state in which there are substantially no three-dimensionally overlapping portions fixed when measured by SEM, preferably 10% or less of the total, more preferably 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Whether or not a single layer is present is preferably determined by measuring multiple points, preferably n=5, more preferably n=10 or more, and calculating the average of the measured points. The determination of whether or not a single layer is present can also be performed by vacuum drying the measurement target, gold sputtering, and setting the conditions to a magnification of 2000x or more. If the determination of whether or not a single layer is present cannot be made using SEM, observation can also be performed using an atomic force microscope. Using the technology disclosed herein, particles can be arranged on a substrate in a single layer.

[0018] As used herein, the term "particle" refers to a basic substance having a structure that serves as a template for forming recesses in a substrate in an analytical sensor. It may be a substance containing a biomolecule or a substance containing a particle core (e.g., a substance containing a particle core and a modifier). Individual particles include substances typically used in analytical sensors. The particle core is not particularly limited as long as it can be used as a template in molecular imprinting, and includes artificially produced inorganic and organic particles. Although particle cores with sizes in the nm range are sometimes referred to as particle cores, this distinction is not strictly made in this disclosure, and it is understood that particle cores do not exclude particles with larger or smaller core structures unless specifically intended to limit the size. For example, inorganic particles include metals, metal oxides, nitrides, fluorides, sulfides, borides, and their composite compounds, as well as hydroxyapatite, and preferably silicon dioxide (silica). Examples of organic particles include, but are not limited to, latex cured products, dextran, chitosan, polylactic acid, poly(meth)acrylic acid, polystyrene, polyethyleneimine, etc. Particles may also be biomolecules or particles having binding functional groups attached to the surface of the particle core. The particle size may vary depending on the intended target in the final analytical sensor, and may include, but is not limited to, about 1 nm to about 100 μm, about 1 nm to about 20 nm, about 20 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 500 nm, about 1 μm to about 10 μm, and about 10 μm to about 100 μm. The particles used in the present disclosure may also be particles in which a modifying substance is integrated into the particle core.

[0019] As used herein, the term "modifier" refers to a substance that directly or indirectly imparts a desired function to an analytical sensor or a substrate for manufacturing an analytical sensor. To impart such functionality, the modifier may contain various functional groups or have a structure capable of incorporating various functional groups within its structure. In the present disclosure, the modifier is advantageously a substance capable of interacting with the particle core, preferably a substance that can be integrated with the particle core through a dissociable interaction without damaging the substrate and / or a substance that can be integrated with the particle core through a dissociable interaction without damaging the particle core. Representative modifiers include, but are not limited to, polyisopropylacrylamide copolymers, poly(meth)acrylamide copolymers, polylactic acid derivatives, poly(meth)acrylic acid copolymers, polymethyl(meth)acrylate copolymers, polyhydroxyethyl(meth)acrylate copolymers, chitosan derivatives, and polylysine derivatives, as well as combinations thereof, as described in detail elsewhere herein. Furthermore, one or more modifiers may be integrated with the particle core.

[0020] As used herein, "integration" generally refers to the formation of a single entity between substances. When referring to a modifier and a particle core, the integration may be achieved by any mechanism that prevents dissociation except under conditions that "allow separation without damaging the particle core" and / or "allow separation without damaging the substrate" in the manufacturing environment of an analytical sensor or a substrate for manufacturing an analytical sensor. Such mechanisms include covalent and non-covalent bonding.

[0021] As used herein, the term "substances used in analytical sensors" refers to substances that are used directly or indirectly in analytical sensors or in the process of producing such sensors, and includes, but is not limited to, substances containing a binding group for a molecule that specifically binds to a target substance, substances containing a reversible linking group, and groups for binding a signal substance.

[0022] As used herein, the term "substrate for preparing an analytical sensor" refers to any substrate for preparing an analytical sensor, and any shape and material can be used as long as they are appropriate for the analytical sensor. Preferably, the substrate for preparing an analytical sensor is a substrate having a recess, and is advantageously configured so that users can easily customize the sensor to be capable of detecting the target substance with even greater sensitivity by modifying the recess with a binding group for a specific binding molecule and a binding group for a signal substance. As used herein, the "substrate for preparing an analytical sensor" is also referred to as a "measurement substrate."

[0023] In this specification, "density" refers to the number of substances present per unit area. For example, 2 is 1mm 2 The density is the number of particles present per unit area. There are various methods for measuring density, but for example, density can be measured by observing the surface with a fluorescence microscope or electron microscope, using the analysis software included with the measuring device, or the particle count function of free image analysis software such as Image J (https: / / imagej.nih.gov / ij / ).

[0024] As used herein, the term "modifier-integrated particle" refers to a particle in which a modifier is integrated into the particle core, and a particle in which a substance having a group for locating a binding group within a molecular imprint recess is integrated with the particle.

[0025] As used herein, the term "group" refers to a monovalent group unless otherwise specified. Examples of non-monovalent groups include alkylene groups (divalent). In addition, when used herein, the term "group" may be omitted in some cases.

[0026] As used herein, the term "reversible linking group" refers to a direct bond or a group with a valency of two or more that is cleavable and reversible. Specific examples include, but are not limited to, the groups shown in 1-3 in Table 1A below.

[0027] [Table 1A-1]

[0028] [Table 1A-2]

[0029] [Table 1A-3]

[0030] As used herein, the term "polymer matrix" refers to a polymer that forms a matrix, typically a material formed by the polymerization of monomers. Advantageously, the matrix may have any shape or structure that is suitable for the sensor when disposed in the analytical sensor or analytical sensor substrate of the present disclosure. The shape or structure of the matrix may be, for example, a thin film or a spherical (particulate) shape. A preferred matrix configuration is one in which the main component is highly biocompatible in order to minimize adsorption of substances other than the target.

[0031] As used herein, the term "molecularly imprinted polymer" refers to any polymer used in molecular imprinting technology (see Takeuchi T. et al. Chromatography, 2016, 37 (2), 43-64), and preferably refers to a substance recognition material having a binding space for a target substance, which is obtained by forming a complex of a target substance or its derivative with a functional monomer through covalent and / or non-covalent bonds, polymerizing it with a crosslinker, and then removing the target substance.

[0032] As used herein, "at least partially compatible with a target" refers to having a shape and / or structure that, when used in an analytical sensor, substantially enables detection or analysis of a target molecule, and when used in the analytical sensor of the present disclosure, refers to a shape that allows interaction of a target molecule within a recess. Without wishing to be bound by theory, if a polymer matrix is ​​formed, there may theoretically be an optimum film thickness that corresponds to half or less of the height of the particle core.

[0033] As used herein, the term "recess" when used in the analytical sensor of the present disclosure refers to a void or hole formed to capture a target, and preferably refers to a void portion formed on a polymer matrix formed in the analytical sensor.

[0034] As used herein, the term "binding group" refers to a group that can bind to a substance. For example, in this specification, a binding group for a signal substance and a binding group for a specific binding molecule are used.

[0035] As used herein, the term "signal substance binding group" refers to a group capable of modifying a signal substance. Examples of signal substances include fluorescent molecules, radioactive element-containing substances, and magnetic substances. From the viewpoint of ease of detection, fluorescent substances are preferred as signal substances. Examples of signal substance binding groups include the binding functional groups listed in 2-1 of Table 2A.

[0036] [Table 2A-1]

[0037] [Table 2A-2]

[0038] As used herein, the term "group for binding a specific binding molecule" refers to a group capable of binding a specific binding molecule that can specifically bind to a target substance. Examples of groups for binding a specific binding molecule to a target substance include the binding functional groups listed in 2-1 of Table 3A.

[0039] [Table 3A-1]

[0040] [Table 3A-2]

[0041] As used herein, "raw materials for a polymer matrix" refers to raw materials that can be reacted to form a polymer matrix. Generally, the formation of a polymer matrix requires at least one monomer and at least one suitable polymerization initiator. However, other components may also be included, such as additional monomers (which may be partially polymerized), additional polymerization initiators, crosslinkers, RAFT agents, catalysts, reducing agents, or solvents. Examples of monomers include, but are not limited to, styrene, N-isopropylacrylamide, and 2-methacryloyloxyethyl phosphorylcholine. Examples of polymerization initiators include, but are not limited to, 2,2'-azobis(isobutyronitrile) (AIBN) and α-ethyl bromoisobutyrate. Examples of crosslinkers include, but are not limited to, melamine compounds, guanamine compounds, glycoluril compounds, N,N'-methylenebisacrylamide, and (tri-, tetra-, penta-, hexa-, or poly)ethylene glycol dimethacrylate. Examples of RAFT agents include, but are not limited to, benzyl benzodithioate, 2-cyano-2-[(dodecylsulfanylthiocarbonyl)sulfanyl]propane, etc. Examples of catalysts include, but are not limited to, CuBr2, etc. Examples of reducing agents include, but are not limited to, ascorbic acid, etc. Examples of solvents are selected without particular limitation from those commonly known as solvents, such as, but not limited to, pure water, buffer solutions, MeOH, EtOH, DMA, and DMF.

[0042] In this specification, the phrase "(the raw materials of) the polymer matrix are polymerized" refers to polymerizing (the raw materials of) the polymer matrix when the raw materials of the polymer matrix are present.

[0043] As used herein, the term "conditions under which particles are dissociated" refers to conditions under which particles disposed on a substrate can be separated from the substrate by severing the interaction between the substrate and the particles. Examples of such conditions include heating, cooling, pH adjustment (acid treatment, alkali treatment), washing with a surfactant-containing solution, ultrasonic irradiation, light irradiation, shaking, and reduction treatment.

[0044] As used herein, the term "substances necessary for analysis" refers to substances capable of capturing and detecting target substances. Examples include capture agents and labels. The capture agent refers to any agent for capturing the substance to be measured, and examples include antibodies, antibody fragments, antibody mimetics, nucleic acid aptamers (including DNA, RNA, peptide nucleic acids, and artificial nucleic acids), phospholipid-recognizing proteins, and lectins.

[0045] As used herein, the term "label" refers to an entity (e.g., substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include the RI (radioisotope) method, the fluorescence method, the biotin method, and the chemiluminescence method. In the present disclosure, when multiple (two or more) markers or factors or means for capturing them are labeled by the fluorescence method, the labeling is carried out with fluorescent substances that have mutually different maximum fluorescence emission wavelengths. The difference in maximum fluorescence emission wavelength is preferably 10 nm or more. When labeling a ligand, any substance that does not affect the function can be used, but examples of fluorescent substances include Alexa TM Examples include Fluor, BODIPY, ATTO, quantum dots (QDots), and fluorescent proteins (GFP, YFP, mCherry, etc.). TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that corresponds to a wide range of fluorescent wavelengths. Compared to other fluorescent dyes of the corresponding wavelength, it is very stable, bright, and has low pH sensitivity. Fluorescent dye combinations with a maximum fluorescence wavelength of 10 nm or more include Alexa TM 555 and Alexa TM 633 combinations, Alexa TM 488 and Alexa TMExamples of suitable labeling methods include the combination of Cy5 and Cy3, Cy5 ...

[0046] As used herein, the phrase "coefficient of variation is XX or less" means that the coefficient of variation in the detection of a target substance by the analytical sensor of the present disclosure is a constant value. The coefficient of variation can be measured by dividing the standard deviation by the average value. In the present disclosure, the coefficient of variation may be 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, with 20% or less being a preferred embodiment. The coefficient of variation may be within a substrate and / or between substrates, or may satisfy both.

[0047] As used herein, "at a rate of YY or more" means that analytical sensors of the present disclosure having performance equal to or greater than a certain value can be produced at a rate of YY or more. Performance in this specification can be measured, for example, using an automatic pipetting device equipped with a fluorescence detector (manufactured by System Instruments Co., Ltd.) or by using the coefficient of variation in target substance detection. In the present disclosure, it is preferable that analytical sensors can be produced at a rate of 80% or more.

[0048] In this specification, "XX to YY pieces / mm on the substrate" 2 "Density of particles or recesses" refers to the density of particles or recesses arranged on the substrate that is between XX and YY / mm 2In this specification, the density is measured using a fluorescent microscope or a scanning electron microscope.

[0049] As used herein, the term "spin coating method" refers to a method in which a particle dispersion liquid in which particles are dispersed in a solvent is added to a substrate using an apparatus that forms a thin film by centrifugal force by rotating a smooth substrate at high speed, and then the substrate is rotated to coat the particles onto the substrate.

[0050] In this specification, the term "method of dropping particles onto a substrate" refers to a method of applying particles onto a substrate by dropping a particle dispersion in which particles are dispersed in a solvent onto the substrate.

[0051] In this specification, the term "method of immersing a substrate in a particle dispersion" refers to a method of coating particles on a substrate by wetting the substrate with a particle dispersion in which particles are dispersed in a solvent.

[0052] In this specification, the term "method of pulling up a substrate from a particle dispersion" refers to a method of coating particles onto a substrate by vertically pulling up the substrate from a particle dispersion in which particles are dispersed in a solvent.

[0053] In this specification, the term "method of spraying a particle dispersion" refers to a method of applying particles to a substrate by spraying a particle dispersion in which particles are dispersed in a solvent onto the substrate.

[0054] As used herein, the term "signal substance" refers to a substance capable of detecting a target substance, and is used synonymously with "label." Examples include fluorescent molecules, substances containing radioactive elements, and magnetic substances. From the viewpoint of ease of detection, fluorescent substances are preferred as signal substances.

[0055] As used herein, the term "specific," when referring to affinity, refers to interacting with a target molecule with higher affinity than with other substances, and preferably refers to not interacting with other substances; the latter term is also referred to as "specific in the narrow sense" or "selectively specific." Specificity can be determined using various molecular interaction analysis devices. For example, measurement is possible using various analytical devices using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or quartz crystal macrobalance (QCM).

[0056] (Preferred embodiment) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0057] <Base material for manufacturing analytical sensors> In one aspect, the present disclosure provides a substrate for fabricating an analytical sensor, comprising: A) a substrate; and B) particles arranged in a monodisperse manner on the substrate. This substrate exhibits excellent properties such as stability, efficiency, performance, and reproducibility due to the substrate being fabricated with the particles arranged in a monodisperse manner.

[0058] In one aspect, the present disclosure provides a substrate for fabricating an analytical sensor, comprising: A) a substrate; and B) particles arranged on the substrate without agglomeration. This substrate exhibits excellent properties such as stability, efficiency, performance, and reproducibility by being manufactured with the particles arranged on the substrate without agglomeration.

[0059] In one aspect, the present disclosure provides a substrate for fabricating an analytical sensor, comprising A) a substrate and B) particles arranged on the substrate in a single layer. This substrate exhibits excellent properties such as stability, efficiency, performance, and reproducibility because it is manufactured with particles arranged in a single layer.

[0060] In another aspect, the present disclosure provides a method for preparing a granular material comprising: dissolving A) a substrate and B) particles in a granular material at a concentration of 1×10 3 pieces / mm 2 ~1×10 6 pieces / mm 2 and particles arranged on the substrate at a density of 1000 to 15000. The substrate exhibits excellent properties such as stability, efficiency, performance, and reproducibility by being manufactured with the particles arranged at a specific density.

[0061] The substrate material used in the present disclosure may be, for example, a material selected from the group consisting of metals, metal oxides, glass, paper (cellulose), cloth, silicon dioxide, silicon, resins, and combinations thereof. Metals include gold, silver, copper, aluminum, titanium, tungsten, molybdenum, etc. Resins include poly(meth)acrylate, polystyrene, ABS (acrylonitrile-butadiene-styrene copolymer), polycarbonate, polyester, polyethylene, polypropylene, nylon, polyurethane, silicone resin, fluororesin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, vinyl chloride resin, etc.

[0062] The substrate used in the present disclosure may be formed by combining multiple materials selected from the above-mentioned materials. For example, the substrate may be a glass or resin substrate having a metal film formed on its surface. The substrate may be in the form of a plate or particles. Preferred examples include gold substrates, glass substrates, gold nanoparticles, silicon dioxide particles (silica particles, glass beads, etc.), etc.

[0063] The particles used in the present disclosure may be biomolecules or particle cores. The particle cores are not particularly limited as long as they can be used as templates for molecular imprinting, and include artificially produced inorganic and organic particles. Inorganic particles include metals (e.g., gold, silver, platinum, tin-doped indium oxide (ITO), antimony-doped tin oxide (ATO)), metal oxides (e.g., iron oxide, aluminum oxide, copper oxide, titanium oxide, zinc oxide, zirconium oxide, cerium oxide, cobalt oxide), graphene, graphene oxide, carbon nanotubes, nanodiamonds, nitrides, fluorides, sulfides, borides, and composite compounds thereof, as well as hydroxyapatite, and preferably silicon dioxide (silica). Examples of organic particles include latex hardeners, dextran, chitosan, polylactic acid, poly(meth)acrylic acid, polymethyl methacrylate, PLGA (poly(lactic-co-glycolic acid)), polystyrene, polyethyleneimine, etc. Furthermore, particles may have binding functional groups attached to the surface of biomolecules or particle cores.

[0064] The particle density on the substrate surface of the present disclosure can be controlled by appropriately adjusting the particle placement technique, the solvent in which the particles are dispersed, the particle concentration, the amount of particle dispersion, the temperature of the particle dispersion, etc. By controlling the particle density on the substrate surface, it becomes possible to control the density of the recesses in the analytical sensor, and an analytical sensor that exhibits excellent properties in terms of stability, efficiency, performance, etc. can be produced.

[0065] In one embodiment, the particles of the present disclosure are preferably about 10 3 pieces / mm 2 ~about 10 8 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 7 pieces / mm 2 , and more preferably about 10 4 pieces / mm 2~about 10 6 pieces / mm 2 The upper limit of the density is, for example, about 1 × 10 7 pieces / mm 2 , about 9×10 6 pieces / mm 2 , about 8×10 6 pieces / mm 2 , about 7×10 6 pieces / mm 2 , about 6×10 6 pieces / mm 2 , about 5×10 6 pieces / mm 2 , about 4×10 6 pieces / mm 2 , about 3×10 6 pieces / mm 2 , about 2×10 6 pieces / mm 2 , about 1×10 6 pieces / mm 2 The lower limit is, for example, about 1 × 10 3 pieces / mm 2 , about 2×10 3 pieces / mm 2 , about 3×10 3 pieces / mm 2 , about 4×10 3 pieces / mm 2 , about 5×10 3 pieces / mm 2 , about 6×10 3 pieces / mm 2 , about 7×10 3 pieces / mm 2 , about 8×10 3 pieces / mm 2 , about 9×10 3 pieces / mm 2 , about 1×10 4 pieces / mm 2 or preferably about 10 3 pieces / mm 2 ~about 10 10 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 9 pieces / mm 2 , and more preferably about 10 4 pieces / mm2 ~about 10 8 pieces / mm 2 The upper limit of the density is, for example, about 1 × 10 10 pieces / mm 2 , about 9×10 9 pieces / mm 2 , about 8×10 9 pieces / mm 2 , about 7×10 9 pieces / mm 2 , about 6×10 9 pieces / mm 2 , about 5×10 9 pieces / mm 2 , about 4×10 9 pieces / mm 2 , about 3×10 9 pieces / mm 2 , about 2×10 9 pieces / mm 2 , about 1×10 9 pieces / mm 2 The lower limit is, for example, about 1 × 10 3 pieces / mm 2 , about 2×10 3 pieces / mm 2 , about 3×10 3 pieces / mm 2 , about 4×10 3 pieces / mm 2 , about 5×10 3 pieces / mm 2 , about 6×10 3 pieces / mm 2 , about 7×10 3 pieces / mm 2 , about 8×10 3 pieces / mm 2 , about 9×10 3 pieces / mm 2 , about 1×10 4 pieces / mm 2 Examples include:

[0066] In one embodiment, the particles used in the present disclosure include a substance used in an analytical sensor. The substance used in the analytical sensor can be a substance necessary for measurement and can be detected. Examples include capture agents and labels. The capture agent refers to any agent for capturing the object to be measured, and examples include antibodies, antibody fragments, antibody mimetics, nucleic acid aptamers (including DNA, RNA, peptide nucleic acids, and artificial nucleic acids), phospholipid-recognizing proteins, and lectins.

[0067] In a preferred embodiment, the particles include particles having modifiers incorporated therein.

[0068] In another aspect, the present disclosure includes, in addition to the particles and substrate, C) a polymer matrix, if necessary, disposed on the substrate body, the polymer matrix having a recess that at least partially fits a target, and the particles are disposed in the recess. It is understood that this analytical sensor fabrication substrate can employ any embodiment described elsewhere in this specification, for example, in the sections <Analytical sensors> and <Method for manufacturing a substrate for analytical sensors>.

[0069] <Analytical sensors> In another aspect, the present disclosure provides an analytical sensor comprising: A) a substrate body; B) optionally, a polymer matrix disposed on the substrate body, the polymer matrix having a recess that at least partially fits a target, and the particles disposed in the recess; C) a binding group for a signal substance disposed on the particles; and D) a binding group for a specific binding molecule that binds to a molecule to be detected disposed on the particles. It is understood that this sensor can employ any embodiment described elsewhere in this specification, for example, in the section on substrates for preparing analytical sensors.

[0070] In one embodiment, the particles used in the present disclosure are 10 3 pieces / mm 2 ~10 8 pieces / mm 2It is preferable that the particles are arranged on the substrate body at a density of about 10 3 pieces / mm 2 ~about 10 7 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 7 pieces / mm 2 , and more preferably about 10 5 pieces / mm 2 ~about 10 6 pieces / mm 2 The upper limit of the density is, for example, about 1 × 10 7 pieces / mm 2 , about 9×10 6 pieces / mm 2 , about 8×10 6 pieces / mm 2 , about 7×10 6 pieces / mm 2 , about 6×10 6 pieces / mm 2 , about 5×10 6 pieces / mm 2 , about 4×10 6 pieces / mm 2 , about 3×10 6 pieces / mm 2 , about 2×10 6 pieces / mm 2 , about 1×10 6 pieces / mm 2 The lower limit is, for example, about 1 × 10 3 pieces / mm 2 , about 2×10 3 pieces / mm 2 , about 3×10 3 pieces / mm 2 , about 4×10 3 pieces / mm 2 , about 5×10 3 pieces / mm 2 , about 6×10 3 pieces / mm 2 , about 7×10 3 pieces / mm 2 , about 8×10 3 pieces / mm 2 , about 9×10 3 pieces / mm 2 , about 1×10 4 pieces / mm2 Or 10 10 pieces / mm 2 It is preferable that the particles are arranged on the substrate body at a density of about 10 3 pieces / mm 2 ~about 10 9 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 8 pieces / mm 2 , and more preferably about 10 5 pieces / mm 2 ~about 10 7 pieces / mm 2 The upper limit of the density is, for example, about 1 × 10 10 pieces / mm 2 , about 9×10 9 pieces / mm 2 , about 8×10 9 pieces / mm 2 , about 7×10 9 pieces / mm 2 , about 6×10 9 pieces / mm 2 , about 5×10 9 pieces / mm 2 , about 4×10 9 pieces / mm 2 , about 3×10 9 pieces / mm 2 , about 2×10 9 pieces / mm 2 , about 1×10 9 pieces / mm 2 The lower limit is, for example, about 1 × 10 3 pieces / mm 2 , about 2×10 3 pieces / mm 2 , about 3×10 3 pieces / mm 2 , about 4×10 3 pieces / mm 2 , about 5×10 3 pieces / mm 2 , about 6×10 3 pieces / mm 2 , about 7×10 3 pieces / mm 2 , about 8×10 3 pieces / mm 2 , about 9×103 pieces / mm 2 , about 1×10 4 pieces / mm 2 If the density is smaller than the range, it is not possible to measure with sufficient sensitivity to detect the target, and if the density is larger than the range, the particles will aggregate, making it difficult to produce a sensor with good reproducibility. A preferred density of monodispersed or non-aggregated recesses is 10 4 pieces / mm 2 ~10 6 pieces / mm 2 Examples include:

[0071] <Base material for producing analytical sensors and analytical sensor manufacturing method> In another aspect, the present disclosure provides a method for producing a substrate for producing an analytical sensor, the method comprising the steps of: A) providing particles of the present disclosure; B) adding the particles to a substrate so that the particles are monodispersely arranged in the substrate body; C) optionally providing a raw material for a polymer matrix to the substrate on which the particles are fixed; D) optionally subjecting the substrate to conditions under which the polymer matrix polymerizes, thereby forming a substrate on which the polymer matrix is ​​disposed; and E) subjecting the substrate to conditions under which the particles dissociate from the substrate, thereby forming recesses.

[0072] In another aspect, the present disclosure provides a method for producing a substrate for producing an analytical sensor, the method comprising the steps of: A) providing particles of the present disclosure; B) adding the particles to a substrate so that the particles are arranged in the substrate body without agglomeration; C) optionally providing a raw material for a polymer matrix to the substrate on which the particles are fixed; D) optionally subjecting the substrate to conditions under which the polymer matrix polymerizes, thereby forming a substrate on which the polymer matrix is ​​disposed; and E) subjecting the substrate to conditions under which the particles dissociate from the substrate, thereby forming recesses.

[0073] In another aspect, the present disclosure provides a method for producing a substrate for producing an analytical sensor, the method comprising the steps of: A) providing particles of the present disclosure; B) adding the particles to a substrate so that the particles are arranged in a monolayer on the substrate body; C) optionally providing a raw material for a polymer matrix to the substrate on which the particles are fixed; D) optionally subjecting the substrate to conditions under which the polymer matrix polymerizes, thereby forming a substrate on which the polymer matrix is ​​arranged; and E) subjecting the substrate to conditions under which the particles dissociate from the substrate, thereby forming recesses.

[0074] In the present disclosure, the step of adding the particles so that they are monodispersed, and / or non-aggregated, and / or arranged in a monolayer is preferably performed so that the particles are monodispersed, and / or non-aggregated, and / or arranged in a monolayer, even after the polymer matrix is ​​polymerized. By providing an appropriate modifying substance and / or setting appropriate conditions, the dispersibility can be appropriately maintained or modified, so that the particles can be maintained in this state even after the polymer matrix is ​​polymerized. Those skilled in the art can appropriately perform this process based on the contents described in the examples and the like in this specification.

[0075] Specifically, in the present disclosure, the step of adding particles so as to be monodisperse and / or not aggregated and / or arranged in a monolayer is performed by adding particles to a substrate in a density of 10×10 0 cells / µL to 2.5 x 10 10 at a concentration of 1.0 x 10 cells / µL, or per surface area of ​​substrate 0 pieces / mm 2 From 2.5 x 10 10 pieces / mm 2 The upper limit of the concentration is, for example, about 1 × 10 10 pcs / μL, approximately 9×10 9 pcs / μL, approximately 8×10 9 pcs / μL, approximately 7×10 9 pcs / μL, approximately 6×10 9 pcs / μL, approximately 5×10 9 pcs / μL, approximately 4×10 9cells / μL, approximately 3×10 9 cells / μL, approximately 2×10 9 cells / μL, approximately 1×10 9 cells / μL, approximately 9×10 8 cells / μL, approximately 8×10 8 cells / μL, approximately 7×10 8 cells / μL, approximately 6×10 8 cells / μL, approximately 5×10 8 cells / μL, approximately 4×10 8 cells / μL, approximately 3×10 8 cells / μL, approximately 2×10 8 cells / μL, approximately 1×10 8 cells / μL, approximately 1×10 8 cells / μL, approximately 9×10 7 cells / μL, approximately 8×10 7 cells / μL, approximately 7×10 7 cells / μL, approximately 6×10 7 cells / μL, approximately 5×10 7 cells / μL, approximately 4×10 7 cells / μL, approximately 3×10 7 cells / μL, approximately 2×10 7 cells / μL, approximately 1×10 7 cells / μL, approximately 9×10 6 cells / μL, approximately​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​0 cells / μL, approximately 4 × 10 0 cells / μL, approximately 5 × 10 0 cells / μL, approximately 6 × 10 0 cells / μL, approximately 7 × 10 0 cells / μL, approximately 8 × 10 0 cells / μL, approximately 9 × 10 0 cells / μL, approximately 1×10 1 cells / μL, approximately 2×10 1 cells / μL, approximately 3 × 10 1 cells / μL, approximately 4 × 10 1 cells / μL, approximately 5 × 10 1 cells / μL, approximately 6 × 10 1 cells / μL, approximately 7 × 10 1 cells / μL, approximately 8 × 10 1 cells / μL, approximately 9 × 10 1 cells / μL, approximately 1×10 2 cells / μL, approximately 2×10 2 cells / μL, approximately 3 × 10 2 cells / μL, approximately 4 × 10 2 cells / μL, approximately 5 × 10 2 cells / μL, approximately 6 × 10 2 cells / μL, approximately 7 × 10 2 cells / μL, approximately 8 × 10 2 cells / μL, approximately 9 × 10 2 cells / μL, approximately 1×10 3 cells / μL, approximately 2×10 3 cells / μL, approximately 3 × 10 3 cells / μL, approximately 4 × 10 3 cells / μL, approximately 5 × 10 3 cells / μL, approximately 6 × 10 3 cells / μL, approximately 7 × 10 3 cells / μL, approximately 8 × 10 3 cells / μL, approximately 9 × 10 3 cells / μL, approximately 1×10 4 cells / μL, approximately 2×10 4 cells / μL, approximately 3 × 10 4 cells / μL, approximately 4 × 10 4 cells / μL, approximately 5 × 10 4 cells / μL, approximately 6 × 10 4 cells / μL, approximately 7 × 10 4 cells / μL, approximately 8 × 10 4 cells / μL, approximately 9 × 10 4 cells / μL, approximately 1×10 5Examples include particles / μL. Unit: mm 2 The upper limit of the amount per substrate having a surface area of ​​about 1 × 10 10 pieces / mm 2 , about 9×10 9 pieces / mm 2 , about 8×10 9 pieces / mm 2 , about 7×10 9 pieces / mm 2 , about 6×10 9 pieces / mm 2 , about 5×10 9 pieces / mm 2 , about 4×10 9 pieces / mm 2 , about 3×10 9 pieces / mm 2 , about 2×10 9 pieces / mm 2 , about 1×10 9 pieces / mm 2 , about 9×10 8 pieces / mm 2 , about 8×10 8 pieces / mm 2 , about 7×10 8 pieces / mm 2 , about 6×10 8 pieces / mm 2 , about 5×10 8 pieces / mm 2 , about 4×10 8 pieces / mm 2 , about 3×10 8 pieces / mm 2 , about 2×10 8 pieces / mm 2 , about 1×10 8 pieces / mm 2 , about 1×10 8 pieces / mm 2 , about 9×10 7 pieces / mm 2 , about 8×10 7 pieces / mm 2 , about 7×10 7 pieces / mm 2 , about 6×10 7 pieces / mm 2 , about 5×10 7 pieces / mm 2 , about 4×10 7 pieces / mm 2 , about 3×107 pieces / mm 2 , about 2×10 7 pieces / mm 2 , about 1×10 7 pieces / mm 2 , about 9×10 6 pieces / mm 2 , about 8×10 6 pieces / mm 2 , about 7×10 6 pieces / mm 2 , about 6×10 6 pieces / mm 2 , about 5×10 6 pieces / mm 2 , about 4×10 6 pieces / mm 2 , about 3×10 6 pieces / mm 2 , about 2×10 6 pieces / mm 2 , about 1×10 6 pieces / mm 2 , about 9×10 5 pieces / mm 2 , about 8×10 5 pieces / mm 2 , about 7×10 5 pieces / mm 2 , about 6×10 5 pieces / mm 2 , about 5×10 5 pieces / mm 2 , about 4×10 5 pieces / mm 2 , about 3×10 5 pieces / mm 2 , about 2×10 5 pieces / mm 2 , about 1×10 5 pieces / mm 2 The lower limit is, for example, about 1 × 10 0 pieces / mm 2 , about 2×10 0 pieces / mm 2 , about 3×10 0 pieces / mm 2 , about 4×10 0 pieces / mm 2 , about 5×10 0 pieces / mm 2 , about 6×10 0 pieces / mm 2 , about 7×100 pcs / mm 2 Approximately 8×10 0 pcs / mm 2 Approximately 9×10 0 pcs / mm 2 Approximately 1×10 1 pcs / mm 2 Approximately 2×10 1 pcs / mm 2 Approximately 3×10 1 pcs / mm 2 Approximately 4×10 1 pcs / mm 2 Approximately 5×10 1 pcs / mm 2 Approximately 6×10 1 pcs / mm 2 Approximately 7×10 1 pcs / mm 2 Approximately 8×10 1 pcs / mm 2 Approximately 9×10 1 pcs / mm 2 Approximately 1×10 2 pcs / mm 2 Approximately 2×10 2 pcs / mm 2 Approximately 3×10 2 pcs / mm 2 Approximately 4×10 2 pcs / mm 2 Approximately 5×10 2 pcs / mm 2 Approximately 6×10 2 pcs / mm 2 Approximately 7×10 2 pcs / mm 2 Approximately 8×10 2 pcs / mm 2 Approximately 9×10 2 pcs / mm 2 Approximately 1×10 3 pcs / mm 2 Approximately 2×10 3 pcs / mm 2 Approximately 3×10 3 pcs / mm 2 Approximately 4×10 3 pcs / mm 2 Approximately 5×10 3 pcs / mm 2 Approximately 6×10 3 pcs / mm 2 Approximately 7×10 3 pcs / mm 2, about 8×10 3 pieces / mm 2 , about 9×10 3 pieces / mm 2 , about 1×10 4 pieces / mm 2 , about 2×10 4 pieces / mm 2 , about 3×10 4 pieces / mm 2 , about 4×10 4 pieces / mm 2 , about 5×10 4 pieces / mm 2 , about 6×10 4 pieces / mm 2 , about 7×10 4 pieces / mm 2 , about 8×10 4 pieces / mm 2 , about 9×10 4 pieces / mm 2 , about 1×10 5 pieces / mm 2 In one embodiment, the upper limit is 1.0×10 10 Pieces / (μL mm 2 ) ~ 1.0 × 10 4 Pieces / (μL mm 2 ) and lower limit: 1.0 × 10 0 Pieces / (μL mm 2 ) ~ 1.0 × 10 4 Pieces / (μL mm 2 ) can be.

[0076] In the present disclosure, the step of disposing particles on a substrate can be carried out by any method, for example, dispersing the particles of the present disclosure in a solution, dropping the solution onto a substrate, and leaving the solution to stand or spin coating the solution to dispose the particles.

[0077] In the present disclosure, the step of providing the raw materials of the polymer matrix to the substrate on which the particles are fixed can be carried out by any method, for example, by adding a polymerizable monomer and providing the polymerizable functional group derived from the particle or the functional monomer and the polymerizable monomer as a substrate.

[0078] In the present disclosure, any material suitable for the intended analysis may be used as the raw material for the polymer matrix. Generally, the raw material for the polymer matrix contains at least one monomer and at least one suitable polymerization initiator to form the polymer matrix. However, other materials may also be included, such as additional monomers (which may be partially polymerized), additional polymerization initiators, crosslinking agents, RAFT agents, catalysts, reducing agents, or solvents. Examples of monomers include, but are not limited to, styrene, N-isopropylacrylamide, and 2-methacryloyloxyethyl phosphorylcholine. Examples of polymerization initiators include, but are not limited to, 2,2'-azobis(isobutyronitrile) (AIBN) and α-ethyl bromoisobutyrate. Examples of crosslinking agents include, but are not limited to, melamine compounds, guanamine compounds, glycoluril compounds, N,N'-methylenebisacrylamide, and (tri-, tetra-, penta-, hexa-, or poly)ethylene glycol dimethacrylate. Examples of RAFT agents include, but are not limited to, benzyl benzodithioate, 2-cyano-2-[(dodecylsulfanylthiocarbonyl)sulfanyl]propane, etc. Examples of catalysts include, but are not limited to, CuBr2, etc. Examples of reducing agents include, but are not limited to, ascorbic acid, etc. Solvents are selected without particular limitation from those commonly known as solvents, and include, but are not limited to, pure water, buffer solutions, MeOH, EtOH, DMA, and DMF, etc. It is understood that any embodiment described elsewhere in this specification, such as the sections <Analytical Sensors> and <Method for Producing a Substrate for Producing an Analytical Sensor>, can be employed for the particles and the process of disposing the particles on a substrate monodispersely and / or without aggregation and / or in a monolayer.

[0079] In the present disclosure, the step of forming a substrate having a polymer matrix disposed thereon by subjecting the substrate to conditions for polymerizing the polymer matrix can be carried out by any method, for example, by adding a polymerizable monomer, synthesizing a molecularly imprinted polymer on a portion of the surface of the particle of the present disclosure using the polymerizable functional group derived from the particle or functional monomer and the polymerizable monomer as a substrate, and the polymerization initiation group as a polymerization initiator, thereby forming a polymer matrix having recesses on the surface of the substrate.

[0080] In the present disclosure, the step of forming recesses by subjecting the substrate to conditions that dissociate the particles from the substrate can be carried out by any process, for example, the reversible linking group can be cleaved to remove the particles of the present disclosure. The examples of the present disclosure demonstrate numerous examples of which are shown in the table below. [Table 11A-1]

[0081] [Table 11A-2]

[0082] [Table 11A-3]

[0083] In another aspect, the present disclosure provides a method for producing an analytical sensor. This method includes the steps of: A) providing particles; B) adding the particles to a substrate so that the particles are monodispersely distributed throughout the substrate; C) optionally providing a polymer matrix material to the substrate on which the particles are immobilized; D) optionally subjecting the substrate to conditions under which the polymer matrix polymerizes, thereby forming a substrate on which the polymer matrix is ​​disposed; E) subjecting the substrate to conditions under which the particles dissociate from the substrate, thereby forming recesses; and F) binding a substance required for analysis to the recesses. It is understood that the particles, the step of distributing the particles monodispersely on the substrate, the conditions under which the polymer matrix polymerizes, and the conditions under which the particles dissociate can be any of the embodiments described elsewhere in this specification, such as in the sections <Analytical Sensors> and <Substrates for Fabricating Analytical Sensors>.

[0084] In another aspect, the present disclosure provides a method for producing an analytical sensor. This method includes the steps of: A) providing particles; B) adding the particles to a substrate so that the particles are disposed within the substrate body without agglomeration; C) optionally providing a polymer matrix precursor to the substrate with the particles immobilized; D) optionally providing a substrate with a polymer matrix disposed thereon by subjecting the substrate to conditions under which the polymer matrix polymerizes; E) forming recesses by subjecting the substrate to conditions under which the particles dissociate; and F) binding a substance required for analysis to the recesses. It should be understood that the particles, the step of disposing the particles on the substrate without agglomeration, the conditions under which the polymer matrix polymerizes, and the conditions under which the particles dissociate can be any of the embodiments described elsewhere in this specification, such as in the sections <Analytical Sensors> and <Substrates for Fabricating Analytical Sensors>.

[0085] In another aspect, the present disclosure provides a method for producing an analytical sensor. This method includes the steps of: A) providing particles; B) adding the particles to a substrate so that the particles are arranged in a monolayer on the substrate body; C) optionally providing a polymer matrix precursor to the substrate on which the particles are immobilized; D) optionally subjecting the substrate to conditions under which the polymer matrix polymerizes, thereby forming a substrate on which the polymer matrix is ​​arranged; E) subjecting the substrate to conditions under which the particles dissociate from the substrate, thereby forming recesses; and F) binding a substance required for analysis to the recesses. It is understood that the particles, the step of disposing the particles in a monolayer on the substrate, the conditions under which the polymer matrix polymerizes, and the conditions under which the particles dissociate can be any of the embodiments described elsewhere in this specification, such as in the sections <Analytical Sensors> and <Substrates for Fabricating Analytical Sensors>.

[0086] In one embodiment, the step of binding a substance required for analysis to the recess can be carried out by any method, for example, by modifying a binding group for a specific binding molecule and a binding group for a signal substance to a cleaved reversible linking group generated in the recess by reduction.

[0087] In one embodiment, the present disclosure provides a method for producing a 2.0×10 7 pieces / mm 2 The analytical sensor of the present disclosure is provided with a method for manufacturing the analytical sensor, in which the analytical sensor is manufactured with a coefficient of variation of a certain reference value (e.g., 20%) or less by forming recesses at a density of: The coefficient of variation may be within a substrate or between substrates, and it is sufficient that at least either one is 20% or less, and it is preferable that both are 20% or less.

[0088] The density of particles in the monodisperse, non-aggregate, and / or monolayer arrangement step is about 10 3 pieces / mm 2 ~about 10 8 pieces / mm 2 and preferably about 10 3 pieces / mm 2~about 10 7 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 7 pieces / mm 2 or about 10 3 pieces / mm 2 ~about 10 10 pieces / mm 2 and preferably about 10 3 pieces / mm 2 ~about 10 9 pieces / mm 2 , more preferably about 10 4 pieces / mm 2 ~about 10 8 pieces / mm 2 In one embodiment, the step of disposing in a monodisperse and / or agglomerated and / or monolayer is carried out by disposing the particles on the substrate in a monodisperse and / or agglomerated and / or monolayer form. 3 pieces / mm 2 ~10 10 pieces / mm 2 The preferred density of particles disposed on the substrate is 10 4 pieces / mm 2 ~10 7 pieces / mm 2 The density here can be confirmed by observing the surface using a fluorescent microscope or a scanning electron microscope.

[0089] In one embodiment, in the step of forming the recesses, the monodispersed or non-agglomerated recesses of the present disclosure are formed in a volume of 10 3 pieces / mm 2 ~10 10 pieces / mm 2 The present invention provides a method for forming recesses on a substrate at a density of 10. 3 pieces / mm 2 ~10 7 pieces / mm 2 The density can be confirmed by observing the surface using a fluorescent microscope or a scanning electron microscope.

[0090] In one embodiment, the step of disposing the particles monodispersely and / or without aggregation and / or in a monolayer can be carried out by any method, and includes, for example, a step of disposing the particles by spin coating, a method of dropping the particles onto a substrate, a method of immersing the substrate in a particle dispersion, a method of lifting the substrate from a particle dispersion, a method of spraying (applying) the particles, or a method of printing with an inkjet printer.

[0091] (Example of analytical sensor manufacturing) The process for manufacturing an analytical sensor can be carried out as follows: Particles of the present disclosure are dispersed in a solution, dropped onto a substrate, and placed by standing or spin coating. A polymerizable monomer is added to provide polymerizable functional groups derived from the particles or functional monomers and the polymerizable monomer as a substrate. A polymerization initiation group is used as a polymerization initiator to synthesize a molecularly imprinted polymer on a portion of the surface of the particles of the present disclosure. This forms a polymer matrix with recesses on the surface of the substrate. The particles of the present disclosure are removed by weakening (or dissociating) the interaction between the particle core and the modifier. The reversible linking groups present (or exposed) in the recesses are modified with binding groups for specific binding molecules and binding groups for signal substances.

[0092] (Application) The analytical sensor of the present disclosure is used for sensing a detection target. More specific applications are determined depending on the type of specific binding group, but can be used for purposes such as diagnosis or treatment monitoring based on, for example, kidney function, liver function, the presence or degree of inflammation, the presence or degree of tumor, etc.

[0093] The analytical sensor of the present disclosure can be used in the following applications:

[0094] In vivo / in vitro imaging of measurement targets and applications to treatment Virus sensors in the human body or the environment and It can be used to analyze food, crops, livestock, etc.

[0095] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0096] The present examples illustrate examples of the preparation and use of the compounds of the present disclosure. For cell microscopy, we used a CKX31 (OLYMPUS, Tokyo, Japan), a KUBOTA2800 (KUBOTA, Tokyo, Japan) centrifuge, a CO2 water jacked incubator (Thermo Fisher Scientific Inc., Massachusetts, USA), a KS-243 autoclave (TOMY SEIKO Co., Ltd., Tokyo, Japan), and a bioclean bench (ORIENTAL GIKEN INC., Tokyo, Japan). For exosome concentration measurement, we used a qNano Gold (Izon Science Ltd., Christchurch, New Zealand). The gold substrate was treated with UV ozone using a UV Ozone Cleaner (BioForce Nanosciences, Inc.), and fluorescence measurements were performed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with an automatic SIC dispenser (SYSTEM INSTRUMENTS Co., Ltd., Tokyo, Japan) and spectroscopic software, Andor SOLIS (Andor Technology Ltd, Belfast, Northern Ireland).

[0097] MALDI-TOF-MS was performed using a MALDI-TOF / MS (MALDI-7090, Shimadzu Corporation), analysis software (MALDI Solutions, Shimadzu Corporation), and calibration was performed using Protein Calibration Standard I (Bruker). Sinapic acid was used as the matrix. CD spectra were obtained using a J-725 circular dichroism spectrometer (JASCO, Tokyo, Japan). pH was measured for buffer preparation using a benchtop pH meter F-52 (HORIBA, Kyoto, Japan). Fluorescence spectra were measured using a Hitachi High-Technologies F-2500 fluorescence spectrophotometer (Tokyo, Japan). Amicon Ultra-4 (10 kDa) ultrafiltration membrane was used for ultrafiltration. Absorption spectra were measured using a Thermo Scientific™ Nano Drop™ One ultramicro UV-Visible spectrophotometer (Thermo Fisher). The average particle size and polydispersity index (PDI) of the prepared particles were measured by dynamic light scattering (DLS) using a Zetasizer Nano-ZS MAL500735 (Malvern, UK) and Zetasizer software was used for analysis. TEM analysis was performed using a transmission electron microscope (JEM-1230, JEOL Ltd.).

[0098] Example 1: Synthesis of Modifiers 1. Experimental Procedure 1-1. Polymer synthesis

[0099] [ka]

[0100] 1-1-1. Synthesis of polymer E2

[0101] [ka]

[0102] The reagents in the recipe shown in Table 1-1 were dissolved in DMF (2 mL), degassed, and purged with argon. The polymerization reaction was then carried out in an oil bath (75 °C) for 24 hours. The reaction solution was cooled and exposed to air to terminate the reaction. The reaction solution was then dropped into diethyl ether to produce a precipitate, which was then collected. This procedure was repeated twice. The collected precipitate was dried in vacuo to obtain polymer (E2-0). (Yield: 100 mg) 100 mg of the resulting polymer was dispersed in a 1 / 1 (v / v) dichloromethane / dioxane solution (10 mL), to which 92 mg (0.5 mmol) of N-succinimidyl methacrylate and 140 μL (1.0 mmol) of triethylamine (TEA) were added, followed by stirring at room temperature for 24 hours. After the reaction, the solvent was removed under reduced pressure using an evaporator, and a small amount of dichloromethane was added to the residue. An excess of hexane was then added, and the resulting precipitate was collected (this procedure was repeated twice). The resulting precipitate was dried in vacuo to obtain polymer E2 (yield: 101 mg). The obtained polymer E2 1 The composition was estimated from H-NMR (in DMSO-d6) (AVANCE-500, Bruker) (Figure 1-1).

[0103] [Table 1-1]

[0104] 1-1-2. Synthesis of polymer E3

[0105] [ka]

[0106] The reagents in the recipe shown in Table 1-2 were dissolved in DMF (2 mL), degassed and replaced with argon, and then the polymerization reaction was carried out in an oil bath (75°C) for 24 hours. After the reaction, the solution was cooled and exposed to air to stop the reaction. The reaction solution was then dropped into diethyl ether to produce a precipitate, which was then collected. This procedure was repeated twice. The collected precipitate was dried in a vacuum to obtain the polymer (yield: 105 mg). This polymer was dispersed in dichloromethane (10 mL), to which 110 mg (0.6 mmol) of N-succinimidyl methacrylate and 140 μL (1.0 mmol) of triethylamine (TEA) were added, followed by stirring at room temperature for 24 hours. An excess of hexane was added to the reaction mixture, and the resulting precipitate was collected (this procedure was repeated twice). The resulting precipitate was dried under vacuum to obtain polymer E3 (yield: 115 mg). The resulting polymer was dispersed in dichloromethane (6 mL), to which 1.0 mL of 4 N HCl in dioxane was added, followed by stirring under ice cooling for 2 hours. The mixture was then further stirred at room temperature overnight. Hexane was added to the reaction mixture, and the resulting precipitate was collected. A small amount of dichloromethane was added to the collected precipitate, followed by an excess of hexane, to wash the precipitate. The collected precipitate was then dried under vacuum (yield: 105 mg). The obtained polymer E3 1 The composition was estimated from H-NMR (in DMSO-d6) (AVANCE-500, Bruker) (Figures 1-2).

[0107] [Table 1-2]

[0108] 1-1-3. Synthesis of polymer E4

[0109] [ka]

[0110] The reagents in the recipe shown in Table 1-3 were dissolved in DMF (2 mL), degassed, and purged with argon. The polymerization reaction was then carried out in an oil bath (75°C) for 24 hours. The reaction solution was cooled and exposed to air to terminate the reaction. The reaction solution was then dropped into diethyl ether to produce a precipitate, which was then collected. This procedure was repeated twice. The collected precipitate was dried in a vacuum to obtain a polymer (yield: 70 mg).

[0111] The resulting polymer was dispersed in dichloromethane (10 mL), to which 1.0 mL of 4N HCl in dioxane was added, and the mixture was stirred under ice cooling for 2 hours. The mixture was then stirred at room temperature overnight. After the reaction, hexane was added to the solution, and the resulting precipitate was collected. A small amount of dichloromethane was added to the collected precipitate, followed by an excess of hexane to wash the precipitate. The collected precipitate was dried in vacuum. The polymer was then dispersed in dichloromethane (10 mL), to which 73 mg (0.4 mmol) of N-succinimidyl methacrylate and 84 μL (0.6 mmol) of triethylamine (TEA) were added, and the mixture was stirred at room temperature for 24 hours. An excess of hexane was added to the reaction solution, and the resulting precipitate was collected (this procedure was repeated twice). The resulting precipitate was dried in vacuum to obtain E4 (yield: 62 mg). For the obtained E4 1 The composition was estimated from H-NMR (in DMSO-d6) (AVANCE-500, Bruker) (Figures 1-3).

[0112] [Table 1-3]

[0113] Synthesis of 1-2-1 polymer F1 The reagents in the recipe shown in Table 1-4 were dissolved in DMF (2 mL), degassed, and purged with argon. The polymerization reaction was then carried out in an oil bath (75°C) for 24 hours. The reaction solution was cooled and exposed to air to terminate the reaction. The reaction solution was then dropped into diethyl ether to produce a precipitate, which was then collected. This procedure was repeated twice. The collected precipitate was vacuum dried to obtain a polymer (yield: 105.7 mg). The resulting polymer was dispersed in dichloromethane (5 mL), to which 34.1 mg (0.19 mmol) of N-succinimidyl methacrylate and 38.9 μL (0.28 mmol) of triethylamine (TEA) were added, followed by stirring at room temperature for 24 hours. An excess of hexane was added to the reaction solution, and the resulting precipitate was collected (this procedure was repeated twice). The resulting precipitate was dried under vacuum to obtain F1 (yield: 19.1 mg). Regarding the F1 obtained 1 The composition was estimated from H-NMR (in DMSO-d) (AVANCE-500, Bruker).

[0114] [Table 1-4]

[0115] Synthesis of polymer (AN1) 64 mg of boc-cystamine methacrylamide, 29 μL of t-butyl acrylate, and 68 mg of NIPAm were dissolved in 1.8 mL of DMF. Finally, 8.6 mg of RAFT agent (2-cyano-2-[(dodecylsulfanylthiocarbonyl)sulfanyl]propane) and 2.1 mg of initiator (AIBN) in 0.2 mL of DMF were added. The mixture was degassed and purged with argon to remove dissolved oxygen. The polymerization reaction was then carried out in an oil bath set at 75°C (24 h). After the reaction, the reaction solution was added dropwise to an excess amount of diethyl ether, and the precipitate was collected (yield: 51 mg). The resulting polymer was dissolved in dichloromethane (5 mL) and stirred under ice cooling. 1.5 mL of trifluoroacetic acid was added and the mixture was stirred overnight in the dark. After the reaction, diethyl ether was added, and the resulting precipitate was collected and washed with diethyl ether. (Yield: 22 mg) The resulting polymer was dispersed in dichloromethane, to which 15 mg (0.08 mmol) of N-succinimydyl methacrylate and 17 μL (0.12 mmol) of triethylamine were added, and the mixture was stirred overnight at room temperature. Diethyl ether was added to the reaction mixture, and the resulting precipitate was collected. This precipitate was dissolved in a small amount of dichloromethane, and an excess amount of hexane was added to precipitate the polymer. This procedure was repeated twice, and the polymer was dried in vacuo to obtain the desired polymer (AN1). (Yield: 16 mg)

[0116] 1-1. Polymer (E101) synthesis [ka] Polymer Recipe: [Table A-1]

[0117] The compounds from the above recipe are placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator are added last, and after adding a stirrer, the atmosphere is degassed and replaced with argon, and polymerization is carried out in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the reaction is stopped by cooling the Schlenk flask on ice and introducing air. The reaction solution is added to a large amount of diethyl ether, and the resulting precipitate is collected. A small amount of CHCl is added to the residue, and an excess amount of n-hexane is added to wash the precipitate, which is then dried in vacuo.

[0118] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 54 mg (0.3 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E101).

[0119] 1-2. Polymer (E101)-particle core complex formation and immobilization on a substrate A polymer (E101) aqueous solution (2 mg / mL) and a particle core dispersion with basic groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80 times with DMF. 4 μL of this solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride and allowed to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0120] 2-1. Polymer (E102) synthesis [ka] Polymer Recipe: [Table A-2]

[0121] The compounds from the above recipe are placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator are added last, and after adding a stirrer, the atmosphere is degassed and replaced with argon, and polymerization is carried out in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the reaction is stopped by cooling the Schlenk flask on ice and introducing air. The reaction solution is then added to a large amount of diethyl ether, and the resulting precipitate is collected. A small amount of CHCl is added to the residue, and an excess amount of n-hexane is added to wash the precipitate, which is then dried in vacuo.

[0122] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 54 mg (0.3 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E102).

[0123] 2-2. Polymer (E102)-particle core complex formation and immobilization on a substrate A 2 mg / mL aqueous solution of polymer (E102) and a dispersion of particle cores bearing hydrophobic groups such as benzene rings on their surfaces were mixed at a 1 / 1 ratio (v / v). This solution was then diluted 80 times with DMF. 4 μL of this solution was then dropped onto a gold substrate modified with SAMs of 2-(2-bromoisobutyryloxy)undecylthiol alone or a mixture of 2-(2-bromoisobutyryloxy)undecylthiol and undecanethiol, and allowed to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0124] 3-1. Polymer (E103) synthesis [ka] Polymer Recipe: [Table A-3]

[0125] The compounds from the above recipe are placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator are added last, and after adding a stirrer, the atmosphere is degassed and replaced with argon, and polymerization is carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the reaction is stopped by cooling the Schlenk flask on ice and introducing air. The reaction solution is added to a large amount of diethyl ether, and the resulting precipitate is collected. A small amount of CHCl is added to the residue, and an excess amount of n-hexane is added to wash the precipitate, which is then dried in vacuo.

[0126] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 54 mg (0.3 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E103).

[0127] 3-2. Polymer (E103)-particle core complex formation and immobilization on a substrate A 2 mg / mL aqueous solution of polymer (E103) and a dispersion of particle cores bearing hydrophobic groups such as benzene rings or alkyl chains on the surface were mixed at a 1 / 1 ratio (v / v). This solution was then diluted 80 times with DMF. 4 μL of this solution was then dropped onto a gold substrate modified with SAM of 2-(2-bromoisobutyryloxy)undecylthiol alone or a mixture of 2-(2-bromoisobutyryloxy)undecylthiol and undecanethiol, and allowed to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0128] 4-1. Polymer (E104) synthesis [ka] Polymer Recipe: [Table A-4]

[0129] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stir bar was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum.

[0130] The polymer prepared in the previous section, TEA (140 μL, 1.0 mmol), was dissolved in CHCl and stirred under ice cooling. 54 mg (0.3 mmol) of N-succinimidyl methacrylate was added and stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E104).

[0131] 4-2. Polymer (E104)-particle core complex formation and immobilization on a substrate A polymer (E104) aqueous solution (2 mg / mL) and a particle core dispersion bearing a coordinate-bonding group such as a His-tag on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80-fold with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol was immersed in CHCl containing 0.1 M EDC and 0.05 M NHS and left to stand for 1 hour. After the reaction, the substrate was washed with CHCl and dried with nitrogen. 4 μL of the polymer (E104)-particle core mixed solution was dropped onto the substrate and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0132] 5-1. Polymer (E105) synthesis [ka] 100 mg of polymer (E101) and 140 μL (1.0 mmol) of TEA were dissolved in CHCl and stirred under ice cooling. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added and the mixture was further stirred overnight. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E101)-NHS (E112). 10 mg of the polymer prepared in the previous section and 28 μL of TEA were dissolved in DMF (1 mL) and stirred. 3 mg of synthetic peptide (six histidines bound from the N-terminus, three glycines bound, and a lysine bound to the C-terminus, with the C-terminal carboxyl group amidated) was added and the mixture was stirred overnight. After the reaction, a small amount of CHCl was added, followed by an excess of n-hexane to produce a precipitate. The precipitate was collected, a small amount of CHCl was added, and an excess of n-hexane was added to wash the precipitate, which was then dried in vacuo to obtain polymer (E105).

[0133] 5-2. Polymer (E105)-particle core complex formation and immobilization on a substrate A polymer (E105) aqueous solution (2 mg / mL) and a particle core dispersion bearing coordinate-bonding groups such as NTA groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80-fold with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride was immersed in 1 mM N-[5-(4-Isothiocyanatobenzyl)amido-1-carboxypentyl]iminodiacetic acid dissolved in DMSO and left to stand for 1 hour. After the reaction, the substrate was washed with DMSO and EtOH and dried with nitrogen. 4 μL of the polymer (E105)-particle core mixed solution was dropped onto the substrate and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0134] 6-1. Polymer (E106) synthesis [ka]

[0135] 100 mg of polymer (E101) and 140 μL (1.0 mmol) of TEA were dissolved in CHCl and stirred under ice cooling. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added and the mixture was further stirred overnight. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E101)-NHS (E112).

[0136] 10 mg of the polymer prepared in the previous section was dissolved in 1 mL of 50 mM carbonate buffer (pH 8.5) and stirred. 3 mg of Avidin was added and the mixture was stirred overnight. The reaction solution was placed in a 100 kDa dialysis membrane and dialyzed against phosphate buffer (pH 7.4). After dialysis, the solution was concentrated by ultrafiltration to obtain a solution of the desired polymer (E106).

[0137] 6-2. Polymer (E106)-particle core complex formation and immobilization on a substrate A polymer (E106) aqueous solution (2 mg / mL) and a particle core dispersion bearing biotin or its derivatives on the surface are mixed at a 1 / 1 ratio (v / v). This solution is then appropriately diluted with phosphate buffer. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride is immersed in DMSO containing 0.1 M EDC and 0.05 M biotin and left to stand for 1 hour. After the reaction, the substrate is washed with DMSO and EtOH and dried with nitrogen. 40 μL of the polymer (E105)-particle core mixed solution is then dropped onto the substrate and left to stand for 1 hour. The substrate is then washed with phosphate buffer to obtain a particle-immobilized substrate.

[0138] 7-1. Polymer (E107) synthesis [ka] Polymer Recipe: [Table A-5]

[0139] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stir bar was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum.

[0140] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 54 mg (0.3 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E107).

[0141] 7-2. Polymer (E107)-particle core complex formation and immobilization on a substrate A polymer (E107) aqueous solution (2 mg / mL) and a particle core dispersion with Avidin functionalized on the surface were mixed at a 1 / 1 ratio (v / v). This solution was then diluted appropriately with PBS. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol was immersed in 0.1 M EDC and 0.05 M NHS in CHCl and left to stand for 1 hour. After the reaction, the substrate was washed with CHCl and dried with nitrogen. A 1 mg / mL Avidin solution was added dropwise to the substrate and left to stand for 1 hour. After the reaction, the substrate was washed with phosphate buffer. A polymer (E107)-particle core mixed solution was added dropwise to the substrate and left to stand for 1 hour. The substrate was then washed with phosphate buffer to obtain a particle-immobilized substrate.

[0142] 8-1. Polymer (E108) synthesis [ka] 100 mg of polymer (E3) and 140 μL (1.0 mmol) of TEA were dissolved in CHCl and stirred under ice cooling. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of 3-mercaptopropionic acid were added and the mixture was further stirred overnight. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E108).

[0143] 8-2. Polymer (E106)-particle core complex formation and immobilization on a substrate A polymer (E108) aqueous solution (2 mg / mL) and a particle core dispersion bearing thiol-reactive groups such as maleimide and pyridyl disulfide groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was then diluted appropriately with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride was immersed in a solution of 0.1 M EDC and 0.05 M 3-maleimidopropionic acid or 3-(2-pyridyldithio)propionic acid in DMF and left to stand for 1 hour. After the reaction, the substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E108)-particle core mixture was then dropped onto the substrate and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0144] 9-1. Polymer (E109) ​​synthesis [ka] 100 mg of polymer (E3) and 140 μL (1.0 mmol) of TEA were dissolved in CHCl and stirred under ice cooling. 40 mg (0.2 mmol) of EDC and 34 mg (0.2 mmol) of 3-maleimidopropionic acid were added and the mixture was further stirred overnight. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E108).

[0145] 9-2. Polymer (E109)-particle core complex formation and immobilization on a substrate A polymer (E109) ​​aqueous solution (2 mg / mL) and a particle core dispersion with thiol groups on the surface are mixed at a 1 / 1, v / v ratio. This solution is then appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride is immersed in DMF containing 0.1 M EDC and 0.05 M 3-mercaptopropionic acid and left to stand for 1 hour. After the reaction, the substrate is washed with EtOH and dried with nitrogen. 4 μL of the polymer (E109)-particle core mixed solution is then dropped onto the substrate and left to stand for 1 hour. The substrate is then washed with EtOH to obtain a particle-immobilized substrate.

[0146] 10-1. Polymer (E110) synthesis [ka] Polymer Recipe: [Table A-6]

[0147] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum.

[0148] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 54 mg (0.3 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E110).

[0149] 10-2. Polymer (E110)-particle core complex formation and immobilization on a substrate A polymer (E110) aqueous solution (2 mg / mL) and a particle core dispersion with amino groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was then diluted appropriately with DMF. The polymer (E110)-particle core mixture was added dropwise to a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6 undecanethiol and allowed to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0150] 11-1. Polymer (E111) synthesis [ka] Polymer Recipe: [Table A-7]

[0151] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum.

[0152] 140 μL (1.0 mmol) of the polymer TEA prepared in the previous section was dissolved in CHCl and stirred under ice cooling. 54 mg (0.3 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo.

[0153] The resulting polymer is dissolved in CHCl and stirred under ice cooling. 1 mL of 4 N HCl in dioxane is added and the mixture is stirred overnight. n-hexane is added to the reaction mixture and the resulting precipitate is collected. A small amount of CHCl is added to the precipitate, and an excess amount of n-hexane is added to wash the precipitate. The precipitate is then dried in vacuo to obtain polymer (E111).

[0154] 11-2. Polymer (E111)-particle core complex formation and immobilization on a substrate A polymer (E111) aqueous solution (2 mg / mL) and a particle core dispersion with aldehyde groups modified on the surface were mixed at a 1 / 1, v / v ratio. This solution was appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6 undecanethiol was immersed in a solution of 0.1 M EDC and 0.05 M 4-formylbenzoic acid in DMF and left to stand for 1 hour. After the reaction, the substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E111)-particle core mixed solution was dropped onto the substrate and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0155] 12-1. Polymer (E112) synthesis [ka] 100 mg of polymer (E101) and 140 μL (1.0 mmol) of TEA were dissolved in CHCl and stirred under ice cooling. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added and the mixture was further stirred overnight. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E112).

[0156] 12-2. Polymer (E112)-particle core complex formation and immobilization on a substrate A polymer (E112) aqueous solution (2 mg / mL) and a particle core dispersion with amino groups on the surface are mixed at a 1 / 1, v / v ratio. This solution is left to stand overnight and then diluted 80 times with DMF. 4 μL of the polymer (E112)-particle core mixed solution is dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and 1-Decanaminium, 10-mercapto-N,N,N-trimethyl-, chloride and left to stand for 1 hour. The substrate is then washed with EtOH to obtain a particle-immobilized substrate.

[0157] 13-1. Polymer (E113) synthesis [ka] Polymer Recipe: [Table A-8]

[0158] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum.

[0159] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 27 mg (0.15 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E113).

[0160] 13-2. Polymer (E111)-particle core complex formation and immobilization on a substrate A polymer (E113) aqueous solution (2 mg / mL) and a particle core dispersion with phenylboronic acid groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was then diluted appropriately with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride was immersed in a solution of 0.1 M EDC and 0.05 M 4-carboxyphenylboronic acid (or 3-fluoro-4-carboxyphneylboronic acid) in DMF and left to stand for 1 hour. After the reaction, the substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E113)-particle core mixture was then dropped onto the substrate and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0161] 14-1. Polymer (E114) synthesis [ka] Polymer Recipe: [Table A-9]

[0162] The compounds from the above recipe are placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator are added last, and after adding a stirrer, the atmosphere is degassed and replaced with argon, and polymerization is carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the reaction is stopped by cooling the Schlenk flask on ice and introducing air. The reaction solution is added to a large amount of diethyl ether, and the resulting precipitate is collected. A small amount of CHCl is added to the residue, and an excess amount of n-hexane is added to wash the precipitate, which is then dried in vacuo.

[0163] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 27 mg (0.15 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E114).

[0164] 14-2. Polymer (E114)-particle core complex formation and immobilization on a substrate A polymer (E114) aqueous solution (2 mg / mL) and a particle core dispersion modified with cis-diol groups on the surface were mixed at a 1 / 1, v / v ratio. This solution was then diluted appropriately with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride was immersed in a DMF solution containing TEA and α-D-mannopyranosylphenyl isothiocyanate and left to stand for 1 hour. After the reaction, the substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E114)-particle core mixed solution was then dropped onto the substrate and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0165] 15-1. Polymer (E115) synthesis [ka] Polymer Recipe: [Table A-10]

[0166] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum.

[0167] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 27 mg (0.15 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E115).

[0168] 15-2. Polymer (E115)-particle core complex formation and immobilization on a substrate A polymer (E114) aqueous solution (2 mg / mL) and a particle core dispersion with amino groups modified on the surface are mixed at a 1 / 1, v / v ratio. This solution is appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride is immersed in DMF containing EDC and 3-(2-pyridyldithio)propionic acid and left to stand for 1 hour. After the reaction, the substrate is washed with EtOH and dried with nitrogen. 4 μL of the polymer (E115)-particle core mixed solution is dropped onto this substrate and left to stand for 1 hour. The substrate is then washed with EtOH to obtain a particle-immobilized substrate.

[0169] 16-1. Polymer (E116) synthesis [ka] Polymer Recipe: [Table A-11]

[0170] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum. The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 27 mg (0.15 mmol) of N-succinimidyl methacrylate was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E116).

[0171] 16-2. Polymer (E116)-particle core complex formation and immobilization on a substrate A polymer (E116) aqueous solution (2 mg / mL) and a particle core dispersion with alkyne groups on the surface were mixed at a 1 / 1 ratio (v / v). Copper sulfate and sodium ascorbate were added and the mixture was stirred for 1 hour. After the reaction, the solution was replaced with pure water by centrifugation. This solution was then appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride was immersed in DMF containing EDC and 4-(3-Butyn-1-yldithio)butanoic acid and left to stand for 1 hour. After the reaction, the substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E116)-particle core mixed solution (containing copper sulfate and ascorbic acid) was added dropwise to the substrate and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0172] 17-1. Polymer (E116) synthesis [ka] Polymer (E112) and 140 μL (1.0 mmol) of TEA were dissolved in CHCl and stirred under ice cooling. 11 mg (0.2 mmol) of propargylamine was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E117).

[0173] 17-2. Polymer (E117)-particle core complex formation and immobilization on a substrate A polymer (E117) aqueous solution (2 mg / mL) and a particle core dispersion with azide groups on the surface were mixed at a 1 / 1 ratio (v / v). Copper sulfate and sodium ascorbate were added and the mixture was stirred for 1 hour. After the reaction, the solution was replaced with pure water by centrifugation. This solution was then appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6 undecanethiol hydrochloride was immersed in DMF containing EDC and 3-[(2-Azidoethyl)dithio]propanoic acid and left to stand for 1 hour. After the reaction, the substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E117)-particle core mixed solution (containing copper sulfate and ascorbic acid) was added dropwise to the substrate and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0174] 18-1. Polymer (E201) synthesis [ka] Polymer Recipe: [Table A-12]

[0175] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum.

[0176] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 25 mg (0.2 mmol) of N-(2-Aminoethyl)methacrylamide was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E201).

[0177] 18-2. Polymer (E201)-particle core complex formation and immobilization on a substrate A polymer (E201) aqueous solution (2 mg / mL) and a particle core dispersion with acidic groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80 times with DMF. 4 μL of this solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0178] 19-1. Polymer (E202) synthesis [ka] Polymer Recipe: [Table A-13]

[0179] The compounds in the above recipe are placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator are added last, and after adding a stirrer, the atmosphere is degassed and replaced with argon, and polymerization is carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the reaction is stopped by cooling the Schlenk flask on ice and introducing air. The reaction solution is then added to a large amount of diethyl ether, and the resulting precipitate is collected. A small amount of CHCl is added to the residue, and an excess amount of n-hexane is added to wash the precipitate, which is then dried in vacuo.

[0180] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added and the mixture was further stirred overnight. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo.

[0181] 10 mg of the polymer prepared in the previous section and 28 μL of TEA were dissolved in DMF (1 mL) and stirred. 3 mg of synthetic peptide (six histidines bound from the N-terminus, three glycines bound, and a lysine bound to the C-terminus, with the C-terminal carboxyl group amidated) was added and the mixture was stirred overnight. After the reaction, a small amount of CHCl was added, followed by an excess of n-hexane to produce a precipitate. The precipitate was collected, to which a small amount of CHCl was added, followed by an excess of n-hexane, to wash the precipitate, which was then dried in vacuo.

[0182] The polymer prepared in the previous section is dissolved in MeOH, and Ni-complexed L-lysine, N2,N2-bis(carboxymethyl)-N6-(2-methyl-1-oxo-2-propen-1-yl)- is added and stirred. CHCl2 is added, and the precipitate is collected and dried in vacuum to obtain polymer (E202).

[0183] 19-2. Polymer (E202)-particle core complex formation and immobilization on substrate A polymer (E202) aqueous solution (2 mg / mL) and a particle core dispersion with acidic groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80 times with DMF. 4 μL of this solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0184] 20-1. Polymer (E203) synthesis [ka] Polymer Recipe: [Table A-14]

[0185] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum.

[0186] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 40 mg (0.2 mmol) of 4-Methacryloyloxybenzoic acid was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E203).

[0187] 20-2. Polymer (E203)-particle core complex formation and immobilization on a substrate A polymer (E203) aqueous solution (2 mg / mL) and a particle core dispersion with acidic groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80 times with DMF. 4 μL of this solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0188] 21-1. Polymer (E204) synthesis [ka] Polymer Recipe: [Table A-15]

[0189] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum. The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 59 mg (0.2 mmol) of 4-(2-Methacrylamidoethylcarbamoyl)-3-fluorophenylboronic acid was added and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E204).

[0190] 21-2. Polymer (E204)-particle core complex formation and immobilization on substrate A polymer (E204) aqueous solution (2 mg / mL) and a particle core dispersion with acidic groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80 times with DMF. 4 μL of this solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0191] 22-1. Polymer (E205) synthesis [ka] Polymer Recipe: [Table A-16]

[0192] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum.

[0193] The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added and the mixture was further stirred overnight. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo.

[0194] 10 mg of the polymer prepared in the previous section was dissolved in 50 mM carbonate buffer (pH 8.5) (1 mL) and stirred. 3 mg of Avidin was added and the mixture was further stirred overnight. The reaction solution was placed in a 100 kDa dialysis membrane and dialyzed against phosphate buffer (pH 7.4). After dialysis, the solution was concentrated by ultrafiltration. The desired polymer (E205) was then obtained by coupling with biotin monomer ((3aS,4S,6aR)-Hexahydro-2-oxo-N-[3-[(1-oxo-2-propen-1-yl)amino]propyl]-1H-thieno[3,4-d]imidazole-4-pentanamide).

[0195] 22-2. Polymer (E205)-particle core complex formation and immobilization on substrate A polymer (E205) aqueous solution (2 mg / mL) and a particle core dispersion with acidic groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80 times with DMF. 4 μL of this solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0196] 23-1. Polymer (E206) synthesis [ka] Polymer Recipe: [Table A-17]

[0197] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum. The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 38 mg (0.2 mmol) of EDC and 73 mg (0.2 mmol) of 4-[4-[1-(Methacryloyloxy)ethyl]-2-methoxy-5-nitrophenoxy]butanoic acid were added and the mixture was stirred overnight in the dark. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. The precipitate was collected and dried in vacuo to obtain polymer (E206).

[0198] 23-2. Polymer (E206)-particle core complex formation and immobilization on substrate A polymer (E206) aqueous solution (2 mg / mL) and a particle core dispersion with acidic groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80 times with DMF. 4 μL of this solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol and left to stand for 1 hour in the dark. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0199] 24-1. Polymer (E207) synthesis [ka] Polymer Recipe: [Table A-18]

[0200] The compounds in the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After degassing and purging with argon, polymerization was carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask is cooled on ice and air is introduced to stop the reaction. The reaction solution is added to a large amount of diethyl ether and the resulting precipitate is collected. A small amount of CH2Cl2 is added to the residue, and then an excess amount of n-hexane is added to wash the precipitate, which is then dried in a vacuum. The polymer prepared in the previous section, 140 μL (1.0 mmol) of TEA, was dissolved in CHCl and stirred under ice cooling. 38 mg (0.2 mmol) of EDC and 92 mg (0.2 mmol) of compound A (see diagram below) were added and stirred overnight in the dark. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain polymer (E207). [ka]

[0201] 24-2. Polymer (E207)-particle core complex formation and immobilization on substrate A polymer (E207) aqueous solution (2 mg / mL) and a particle core dispersion with acidic groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80 times with DMF. 4 μL of this solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol and left to stand for 1 hour in the dark. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0202] 25-1. Polymer (E301-E312) synthesis [ka] Polymer Recipe: [Table A-19]

[0203] The compounds from the above recipe are placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator are added last, and after adding a stirrer, the atmosphere is degassed and replaced with argon, and polymerization is carried out in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the reaction is stopped by cooling the Schlenk flask on ice and introducing air. The reaction solution is added to a large amount of diethyl ether, and the resulting precipitate is collected. A small amount of CHCl is added to the residue, and an excess amount of n-hexane is added to wash the precipitate, which is then dried in vacuo.

[0204] The prepared polymer, TEA (140 μL, 1.0 mmol), was dissolved in CHCl and stirred under ice cooling. N-succinimidyl methacrylate (36 mg, 0.2 mmol) was added and stirred overnight. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl and an excess of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo.

[0205] The polymer is dissolved in CHCl and stirred under ice cooling. 1 mL of 4 N HCl in dioxane is added and the mixture is stirred overnight. n-hexane is added to the reaction mixture and the resulting precipitate is collected. A small amount of CHCl is added to the precipitate, and an excess amount of n-hexane is added to wash the precipitate, which is then dried in vacuo.

[0206] The polymer and 140 μL (1.0 mmol) of TEA are dissolved in CHCl and stirred on ice. 0.25 mmol of one of the compounds in the table on the right and 20 mg (0.1 mmol) of EDC are added and stirred overnight. After the reaction, n-hexane is added to the reaction solution and the resulting precipitate is collected. This precipitate is redissolved in a small amount of CHCl and an excess amount of n-hexane is added to produce a precipitate. This precipitate is collected and dried in vacuo to obtain the desired polymers (E301-E312). *Used when synthesizing E304-E309 and E311-E312 [Table A-20]

[0207] 25-2. Polymer (E301)-particle core complex formation and immobilization on substrate A polymer (E301) aqueous solution (2 mg / mL) and a particle core dispersion with acidic groups on the surface were mixed at a 1 / 1 ratio (v / v). This solution was diluted 80 times with DMF. 4 μL of this solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol and left to stand for 1 hour. The substrate was then washed with EtOH to obtain a particle-immobilized substrate.

[0208] (Example 1-2: Binding of polymer to particles (non-covalent binding)) 1-2-1. Synthesis of E3-conjugated silica NPs

[0209] [ka]

[0210] Silica nanoparticle dispersion (2.0 × 10 12 100 μL of 1,3,5-triazin-2-yl-4-methylmorpholinium chloride (100 μL / mL), 500 μL of polymer E3 aqueous solution (2.0 mg / mL), and 400 μL of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) aqueous solution (12.5 mM) were mixed and stirred in a thermoshaker at 25 °C and 1000 rpm for 12 hours. After the reaction, the reactants were removed from the solution by centrifugation (25 °C, 10,000 g, 15 min) × 5 times. The supernatants were analyzed by UV-vis measurement (NanoDrop).

[0211] Following the same procedure, polymers E2 and E4 were integrated with silica particles.

[0212] (result) 1-2-2. DLS and Z-potential measurements Silica nanoparticle dispersion (2.0 × 10 12 20 μL of a solution of 2.0 mg / mL of cationic polymers, 100 μL of an aqueous solution of each cationic polymer (2.0 mg / mL), and 80 μL of pure water were mixed and stirred thoroughly using a vortex. This solution was diluted 100 times with pure water to prepare the sample for DLS and Z-potential measurements. The measurement equipment used was a Zetasizer Pro (Marvern Panalytical, UK).

[0213] The results of particle size measurement by DLS (Figure 1-4 and Table 1-5) and Z-potential measurement (Figure 1-5 and Table 1-5) showed that the prepared cationic polymer can be adsorbed onto silica nanoparticles and change their surface properties from a negatively charged state to a positively charged state. Furthermore, no significant change in particle size was observed before and after polymer adsorption onto these silica nanoparticles, indicating that the silica nanoparticles maintain their dispersibility even after polymer adsorption.

[0214] [Table 1-5]

[0215] (Example 1-3: Binding of polymer to particles) Synthesis of polymer (E2-0)-silica nanoparticle composite (RM) [ka] 4.0×10 silica nanoparticles (200 nm, COOH terminal) in pure water (500 μL) 11 The mixture was mixed to a concentration of 2.0 mg / mL of E2-0, 2.0 mg / mL of E2-0, and 10 mM of DMT-MM, and then shaken in a thermoshaker at 25°C, 1,000 rpm, and 15 hours. After the reaction, 500 μL of pure water was added, and the particles were precipitated by centrifugation (25°C, 1,000 rpm, 15 minutes). 900 μL of the supernatant was removed. This procedure was repeated five times to purify the particles. DLS measurements of the particle size and surface charge of the resulting particles are shown in Figures 1-6. The average particle size (Z-Average) was 232 nm (pdi: 0.022) and the surface charge (Z-potential) was +55.3 mV. This confirmed that the polymer had been modified by maintaining the monodispersity of the particles while significantly changing the surface charge to positive.

[0216] (Example 2: Preparation of measurement substrate)

[0217] [ka]

[0218] 2-1. Experiment 2-1-1. Surface modification of gold substrate A gold substrate (9.8 mm x 4.3 mm) was rinsed with EtOH and then treated with UV-O3 for 15 minutes to clean the surface. A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol in a 1:1 ratio in EtOH solvent. The gold substrate was then immersed in this solution. After standing at 30°C for 20 hours, the substrate was washed with EtOH and purified water, allowing a self-assembled monolayer (SAM) to form on the gold substrate surface. 2-1-2. Preparation of particle-immobilized substrate Silica nanoparticle dispersion (particle size 200nm, particle concentration: 2.0×10 11 The complex was formed by mixing polymer E2 (1.0 mg / mL) and polymer E2 (1.0 mg / mL) in water at the aforementioned final concentrations.

[0219] The above complex solution was diluted with DMF, and added dropwise (4 μL) to the substrate surface to give the number of complexes shown in Table 2-2, and allowed to stand at 25° C. for 1 hour. The substrate was washed with DMF, EtOH, and pure water in that order to obtain a particle-immobilized substrate.

[0220] The substrate was evaluated using a fluorescence microscope (KEYENCE, BZ-800) and analyzed using software (Hybrid Cell Count). The measurement conditions are shown in Table 2-1.

[0221] [Table 2-1]

[0222] 2-1-3. Synthesis of silica nanoparticle-imprinted polymer on particle-immobilized substrate A prepolymer solution was prepared with the composition shown in Table 2-2, and after removing dissolved oxygen by freeze degassing, polymerization was carried out in a glove box at 25°C for 18 hours. After polymerization, the substrate was washed with pure water.

[0223] The substrate was immersed in a 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (methanol:pure water = 1:1) and ultrasonicated for 5 minutes to remove the silica nanoparticles and form pores on the surface of the polymer thin film.

[0224] [Table 2-2]

[0225] 2-2.Results 2-2-1. Preparation of particle-immobilized substrate The results of observation using a fluorescence microscope are shown in Figure 2-1. Under all conditions, fluorescence originating from silica nanoparticles was confirmed on the substrate, confirming that silica nanoparticles (polymer composites) were immobilized on the substrate. Figure 2-2 shows the results of analysis using a 60x objective lens for fluorescence observation and hybrid cell counting. Figure 2-2 shows that the number of silica nanoparticles on the substrate tends to increase as the number of droplets increases. Furthermore, in Figures 4 and 5, which clearly show that the nanoparticles are monodispersed, not aggregated, and are in a single layer, the area of ​​the bright spot is 2 μm. 2 Because there was almost nothing that surpassed If the area of ​​the bright spots exceeds this value, it can be determined that the aggregates are aggregates. 2 No. 4 (3 × 10 6 Aggregation occurs when the number of particles is more than 1 × 10 6 It was possible to objectively express numerically that no aggregation occurred when the number of droplets dropped onto the substrate was less than 4 μL, and it was also possible to control the particle state on the substrate by the number of droplets dropped onto the substrate.

[0226] (Example 3: Preparation of measurement substrate (cationic polymer)) 3. Study using silica particles (effect of cationic polymer composition) [ka]

[0227] 3-1. Experiment 3-1-1. Surface modification of gold substrate After rinsing the gold substrate (9.8 mm × 4.3 mm) with EtOH, it was treated with UV-O3 for 15 min to clean up the substrate surface.

[0228] A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol in a 1:1 ratio in EtOH solvent, and the gold substrate was immersed in this solution. After leaving it at 30°C for 20 hours, the substrate was washed with EtOH and pure water, and a self-assembled monolayer (SAM) was formed on the surface of the gold substrate. 3-1-2. Preparation of particle-immobilized substrate Silica nanoparticle dispersion (particle size 200nm, particle concentration: 2.0×10 11 The complexes were formed by mixing the polymers E4 and E3 (1.0 mg / mL) with the polymers E4 and E3 (1.0 mg / mL) in water at the aforementioned final concentrations.

[0229] The above complex solution was diluted with DMF, and the solution (4 μL) was added dropwise to the substrate so that the area of ​​the substrate on which the complex was to be immobilized satisfies Table 3-1 or Table 3-2, and the substrate was allowed to stand at 25° C. for 1 hour.

[0230] The substrate was washed with DMF, EtOH, and pure water in that order to obtain a particle-immobilized substrate.

[0231] The substrate was evaluated using a fluorescence microscope (KEYENCE, BZ-800) and analyzed using software (Hybrid Cell Count). The measurement conditions are shown in Tables 3-1 and 3-2.

[0232] [Table 3-1]

[0233] [Table 3-2]

[0234] 3-2.Results 3-2-1. Preparation of particle-immobilized substrate The results of observation using a fluorescence microscope are shown in Figures 3-1 and 3-2. These results demonstrate that the particle morphology on the substrate can be controlled by the number of droplets dropped onto the substrate, even with different polymer compositions.

[0235] (Example 4: Preparation of measurement substrate (glass substrate)) 4. Study using silica particles (effect of substrate)

[0236] [ka]

[0237] 4-1-1. Surface modification of glass substrates After rinsing the glass substrate (9.8 mm × 4.3 mm) with EtOH, it was treated with UV-O3 for 15 min to clean up the substrate surface.

[0238] 3-Aminopropyltriethoxysilane (ATPES) and 3-(trimethoxysilyl)propyl 2-bromo-2-methylpropanoate were mixed at 1 vol% each in an EtOH solvent (1% pure water), and the glass substrate was immersed in this solution. After standing at 25°C for 1 hour, the glass substrate was washed with EOH and dried in an oven at 90°C for 2 hours, introducing amino groups and ATRP initiating groups onto the surface of the glass substrate.

[0239] A 5 mg / mL succinic anhydride solution (solvent = THF:TEA = 95:5) was prepared, and the above-mentioned substrate was immersed in it. After leaving it at 25°C for 4 hours, it was washed with pure water to convert the amino groups on the glass substrate surface to carboxyl groups.

[0240] The substrate was immersed in a 1 mM 2-bromoisobutanoic acid N-hydroxysuccinimide ester solution (solvent: DMF) and reacted at 25°C for 1 hour, followed by rinsing with EtOH, yielding a glass substrate bearing ATRP initiating groups and carboxyl groups on its surface. 4-1-2. Preparation of particle-immobilized substrate Silica nanoparticle dispersion (particle size 200nm, particle concentration: 2.0×10 11 The polymer E2 (1.0 mg / mL) and polymer E3 (1.0 mg / mL) were mixed in water at the aforementioned final concentrations to form a complex.

[0241] The above complex solution was diluted with DMF, and the solution (4 μL) was added dropwise to the substrate so that the area of ​​the substrate on which the complex was to be immobilized was as shown in Table 4-1, and the substrate was allowed to stand at 25° C. for 1 hour.

[0242] The substrate was washed with DMF, EtOH, and pure water in that order to obtain a particle-immobilized substrate.

[0243] The substrate was evaluated using a fluorescence microscope (KEYENCE, BZ-800) and analyzed using software (Hybrid Cell Count). The measurement conditions are shown in Table 4-1. [Table 4-1]

[0244] 4-2.Results 4-2-1. Preparation of particle-immobilized substrate The results of observation using a fluorescence microscope are shown in Figure 4. These results demonstrate that the particle state on the substrate can be controlled by the number of droplets dropped onto the substrate, even for different substrates.

[0245] (Example 5: Preparation of measurement substrate (polystyrene particles)) 5. Study using polystyrene particles 5-1. Experiment 5-1-1. Surface modification of gold substrate After rinsing the gold substrate (9.8 mm × 4.3 mm) with EtOH, it was treated with UV-O3 for 15 min to clean up the substrate surface.

[0246] A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol in a 1:1 ratio in EtOH solvent, and the gold substrate was immersed in this solution. After leaving it at 30°C for 20 hours, the substrate was washed with EtOH and pure water, and a self-assembled monolayer (SAM) was formed on the surface of the gold substrate. 5-1-2. Preparation of particle-immobilized substrate Polystyrene particle dispersion (particle size 250 nm, particle concentration: 2.0 × 10 11 The complex was formed by mixing polymer E2 (1.0 mg / mL) and polymer E2 (1.0 mg / mL) in water at the aforementioned final concentrations.

[0247] The above complex solution was diluted with DMF, and added dropwise (4 μL) to the substrate area where the complex was to be immobilized, as shown in Table 5-1, and allowed to stand at 25° C. for 1 hour.

[0248] The substrate was washed with DMF, EtOH, and pure water in that order to obtain a particle-immobilized substrate.

[0249] The substrate was evaluated using a fluorescence microscope (KEYENCE, BZ-800) and analyzed using software (Hybrid Cell Count). The measurement conditions are shown in Table 5-1.

[0250] [Table 5-1]

[0251] 5-2.Results 5-2-1. Preparation of particle-immobilized substrate The results of observation using a fluorescence microscope are shown in Figures 5-1 and 5-2. Under all conditions, fluorescence from the polystyrene particles was confirmed on the substrate, confirming that the polystyrene particles (polymer composite) were immobilized on the substrate. The obtained fluorescence microscope images were analyzed using software (Hybrid Cell Count), and the results are shown in Table 5-2. The analysis showed that the number of polystyrene particles on the substrate tended to increase as the number of particles dropped increased, and that the number of bright spots decreased under conditions with a particularly large number of drops (No. 1 and No. 2) compared to No. 3, indicating an increase in the number of aggregates. Furthermore, No.1 (1 × 10 8 No. 2 (3 × 10 7 No. 8 (3 × 10 particles / 4 μL) showed a high degree of particle aggregation on the substrate and poor uniformity, while No. 9 (3 × 10 particles / 4 μL) showed a high degree of particle aggregation on the substrate and poor uniformity. 4 pcs / 4μL) and No.9 (1×10 4 The amount of immobilized polystyrene particles is extremely small in No. 3 (1 × 10 particles / 4 μL). 7 pieces / 4μL)~No.7(1×10 5 To conduct a detailed study of the cells (cells / 4 μL), analysis was performed using fluorescence observation with a 60x objective lens and hybrid cell counting. The results are shown in Figure 5-3 and Figure 5-4, respectively. Figure 5-3 shows that the maximum bright spot area increases with the number of droplets added, indicating that the scale of aggregation is increasing. In addition, in samples No. 6 and No. 7, which are clearly monodisperse and not aggregated, and can be determined to be a single layer, the bright spot area is 2 μm 2Therefore, bright spot areas exceeding this value can be judged to be aggregates. 2 4 μm 2 10μm 2 The number of bright spots above the boundary value when the boundary value was set as 2 μm is shown in Figure 5-4. 2 No. 4 (3 × 10 6 Aggregation occurs when the number of particles is more than 1 × 10 6 No aggregation occurred when the number of droplets dropped onto the substrate was less than 4 μL, and the particle state on the substrate could be controlled by the number of droplets dropped onto the substrate.

[0252] [Table 5-2]

[0253] (Example 6: Performance Test (Evaluation of Particle Density on Substrate)) Density of recesses on 6A substrate Study using fluorescent cationic polymers

[0254] [ka]

[0255] 6-1.Experiment 6-1-1. Surface modification of gold substrate After rinsing the gold substrate (9.8 mm × 4.3 mm) with EtOH, it was treated with UV-O3 for 15 min to clean up the substrate surface.

[0256] A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol in a 1:1 ratio in EtOH solvent, and the gold substrate was immersed in this solution. After leaving it at 30°C for 20 hours, the substrate was washed with EtOH and pure water, and a self-assembled monolayer (SAM) was formed on the surface of the gold substrate. 6-1-2. Preparation of particle-immobilized substrate Silica nanoparticle dispersion (particle size 200nm, particle concentration: 2.0×10 11 The complex was formed by mixing polymer F1 (1.0 mg / mL) and polymer F2 (1.0 mg / mL) in water at the aforementioned final concentrations.

[0257] The above complex solution was diluted with DMF, and added dropwise (4 μL) to the substrate area where the complex was to be immobilized, as shown in Table 6-1, and allowed to stand at 25° C. for 1 hour.

[0258] The substrate was washed with DMF, EtOH, and pure water in that order to obtain a particle-immobilized substrate.

[0259] The substrate was evaluated using a fluorescence microscope (KEYENCE, BZ-800) and analyzed using software (Hybrid Cell Count). The measurement conditions are shown in Table 6-1.

[0260] [Table 6-1]

[0261] 6-1-3. Synthesis of silica nanoparticle-imprinted polymer on particle-immobilized substrate A prepolymer solution was prepared with the composition shown in Table 6-2, and after removing dissolved oxygen by freeze degassing, polymerization was carried out in a glove box at 25°C for 18 hours. After polymerization, the substrate was washed with pure water.

[0262] The substrate was immersed in a solution of methanol and pure water (1:1) and subjected to ultrasonic treatment for 0.5 minutes to remove the silica nanoparticles and form pores on the surface of the polymer thin film.

[0263] [Table 6-2]

[0264] 6-2.Results 6-2-1. Preparation of particle-immobilized substrate The particle size of the composite of silica nanoparticles and fluorescent cationic polymer F1 was measured using a Zeta Sizer. The results of the surface Z potential measurements are shown in Table 6-3. The fact that the surface Z potential changed from negative to positive before and after complex formation confirmed that silica nanoparticles and fluorescent cationic polymer F1 had formed a complex, and the particle size and PDI confirmed that the particles had not aggregated and that their dispersibility had not been impaired.

[0265] [Table 6-3]

[0266] The results of observation using a fluorescence microscope are shown in Figure 6-1. Green fluorescence from the silica nanoparticles and red fluorescence from the fluorescent cationic polymer were confirmed in all samples, confirming that the silica nanoparticle-fluorescent cationic polymer composite was immobilized on the substrate.

[0267] 6-2-2. Confirmation of fluorescent cationic polymer F1 after removing silica nanoparticles Figures 6-2 and 6-3 show the results of observations using a fluorescence microscope after polymerizing the polymer layer on the substrate after immobilizing the silica nanoparticle-fluorescent cationic polymer complex, and after removing the silica nanoparticles after polymerization, respectively. As shown in Figure 6-2, fluorescence from the silica nanoparticles and fluorescent cationic polymer could be confirmed on the substrate even after polymerization, but as shown in Figure 6-3 after the silica nanoparticles were removed, the green fluorescence from the silica nanoparticles could no longer be confirmed, while red fluorescence from the fluorescent cationic polymer could be confirmed. This confirmed that the fluorescent cationic polymer remained in the polymer layer prepared on the substrate (in the pores after the silica nanoparticles were removed) even after the silica nanoparticles were removed by experimental manipulation.

[0268] In addition, since the red fluorescence remaining on the substrate after removing the silica particles comes from the cationic polymer present in the pores after removing the silica particles, the pore density on the substrate was calculated as the number of bright spots of this red fluorescence = the number of pores. As a result, the pore density formed on the substrate under these experimental conditions correlated with the number of silica particles dropped onto the substrate, and was 10 3 ~10 6 pieces / mm 2 This indicates that it is possible to control not only the particle state on the substrate but also the pore density after removing the silica particles by changing the number of droplets dropped onto the substrate.

[0269] 6.B Particle density on substrate The particles on the measurement substrates obtained in Examples 2, 3, and 5 were analyzed using a 60x objective lens for fluorescence observation and a hybrid cell count. Figures 3-1, 3-2, and 5-1 show that the number of silica nanoparticles on the substrate tends to increase as the number of droplets increases. Furthermore, in No. 4 and No. 5, which are clearly monodisperse and not aggregated, and can be judged to be a single layer, the area of ​​the bright spot is 2 μm. 2 Since there were almost no bright spot areas exceeding this, it can be determined that those with a bright spot area exceeding this are aggregates. By using this value as the boundary value, the boundary value of 2 μm 2 No. 4 (3 × 10 6 Aggregation occurs when the number of particles is more than 1 × 10 6 It was possible to objectively express numerically that no aggregation occurred when the number of droplets dropped onto the substrate was less than 1×10 particles / 4μL, and it was also possible to control the particle state on the substrate by changing the number of droplets dropped onto the substrate. The results of analyzing the particles present on the measurement substrate are summarized in Figure 6-4. No. 5 (1×10 particles / 4μL) where no aggregation occurred 6 When particles are spread at a concentration of 1×10 particles / 4μL or less, the 5 pieces / mm 2 When the particles were present at the following particle densities, they were monodispersed, not aggregated, and arranged in a single layer on the substrate.

[0270] (Example 7: Complexation of His-Tag polymer and silica nanoparticles) [ka] [Table B] The compounds from the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last. After adding a stirrer, the atmosphere was degassed and replaced with argon, and polymerization was carried out in an oil bath (set temperature: 70 °C) for 17 h (stirring speed: 300 rpm). After the polymerization, the Schlenk flask was cooled with ice and air was introduced to stop the reaction. The reaction solution was added to a large amount of diethyl ether, and the resulting precipitate was collected. The precipitate was washed with diethyl ether and dried in vacuo. 5 mg of the resulting polymer, His-tag (six histidines bound from the N-terminus, three glycines bound, and a lysine bound to the C-terminus, with the carboxyl group at the C-terminus amidated) 4 mg of PEG-3-methacrylamide, 3.5 mg of pyridyldithioethyl-PEG3-methacrylamide, and 2.8 μL of TEA were dissolved in 0.3 mL of DMF and stirred for 9 hours. CHCl and n-hexane were added to the reaction mixture, and the resulting precipitate was collected and dried under vacuum to obtain the polymer (E11His). iii) E11His A 4 mM NiCl2 aqueous solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and NTA-SAM formation reagent, and after leaving it at room temperature for 15 min, the substrate was washed with pure water. A mixed solution of silica nanoparticles (200 nm, COOH-terminated) and E11His aqueous solution (silica concentration 2.0 × 10 11 The polymer solution (particles / mL, polymer concentration 1 mg / mL) was diluted 4,400-fold with DMF. 4 μL of this solution was dropped onto the gold substrate after reaction with NiCl2 and left to stand for 1 hour. The surface was then washed with EtOH and observed under a fluorescence microscope. The results are shown in Figure 7.

[0271] (Example 8: Cationic silica nanoparticles + anionic polymer Ex8) 1. Synthesis of anionic polymer Ex8 [ka] The compounds in the recipe in the table were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). The RAFT agent and initiator were added last, and a stirrer was added. After the mixture was degassed and replaced with argon, polymerization was carried out in an oil bath (set temperature: 70 °C) for 16 h. After the polymerization, the Schlenk flask was cooled with ice and air was introduced to stop the reaction. The reaction solution was added to a large amount of diethyl ether, and the resulting precipitate was collected. The collected precipitate was washed with diethyl ether and then dried in vacuum to obtain a polymer. The resulting polymer and 70 μL (0.5 mmol) of TEA were dissolved in CHCl and stirred under ice cooling. 54 mg (0.3 mmol) of N-succinimidyl methacrylate were added and stirred overnight. After that, n-hexane was added to the reaction solution and the resulting precipitate was collected. This precipitate was redissolved in a small amount of CHCl, and an excess amount of n-hexane was added to produce a precipitate. This precipitate was collected and dried in vacuo to obtain the desired polymer (Ex8). Yield: 120 mg [Table C] 2. Composite of silica nanoparticles and polymer 1.98 mg (39.6 μL) of silica (red-plane, 200 nm, 50 mg / mL) was mixed with 60.4 μL of pure water (total 100 μL) and added to 800 μL of EtOH in a 1.5 mL Eppendorf tube. Then, 100 μL of 2M HCl and 15 μL of APTES were added and the mixture was incubated at 25°C in a thermoshaker at 1000 rpm for 20 hours. After the reaction, the mixture was centrifuged and washed with ethanol (5000 rpm, 10 min x 3). The prepared silica nanoparticle dispersion (NH2 terminal, 200 nm, 4.0 × 10 11 20 μL of Ex8 carbonate buffer solution (2 mg / mL) was mixed with 20 μL of 50 mM carbonate buffer (pH 8.0) and left to stand at 25°C for 15 minutes. 3.DLS measurement The complex of 2 was diluted 100 times with pure water, and the particle size and Z-potential were measured using DLS. 4. Immobilization on the substrate The complex of 2 was diluted 80-fold with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol:amino-EG6undecanethiol (1:1 vol) and allowed to stand at 25 °C for 1 h. The surface was then washed with EtOH and observed by fluorescence microscopy and SEM (Figure 8). [ka] The results of particle size measurement by DLS and Z-potential measurement suggested that the anionic polymer was adsorbed onto the silica nanoparticles, changing the surface charge from a positive to a negative state. Furthermore, the lack of a significant change in particle size indicated that the particles maintained their dispersibility even after polymer adsorption. [Table D]

[0272] (result) From the observation results in Figure 8, it was observed that the fluorescent spots derived from the silica nanoparticles were monodisperse, not aggregated, and distributed in a single layer. This indicates that even when anionic polymer Ex8 and cationic silica nanoparticles are combined, the particles are monodisperse, not aggregated, and can be immobilized to the substrate in a single layer, just like in the case of cationic polymer Ex8 and anionic silica nanoparticles.

[0273] Example 9: Cationic Nanoparticles + Anionic Polymer [ka]

[0274] (Silica nanoparticles used) · E2-0 conju red-COOH silica (RM) 200 nm 4.0x10 11 pieces / ml

[0275] (polymer solution) Final polymer concentration: 0.87 mg / mL (10 mM PB, pH 7.47)

[0276] (anionic silica) The silica nanoparticle dispersion and polymer AN-1 were mixed to form a complex. (Final silica concentration: 1.0x10 11 pcs / ml, final polymer concentration 0.435mg / mL (5mM PB) The above complex solution was diluted 4, 40, 400, and 4000 times with DMF, and 4 μL was dropped onto the substrate on which the complex was to be immobilized, followed by leaving the substrate to stand for 1 hour at 25° C. The substrate was washed with ethanol to obtain a particle-immobilized substrate.

[0277] (polymer matrix synthesis) Thin film formation by ATRP (25℃, 20 h)

[0278] (Particle core removal source) 300 μL of 50% acetic acid / MeOH was placed in a 1.5 mL tube, and each substrate was immersed in the solution. After ultrasonic treatment for 8 minutes, the substrates were washed with pure water.

[0279] (SS reductive cleavage) The substrate was placed in a 50 mM tris(2-carboxyethyl)phosphine hydrochloride 50% MeOH aqueous solution and reacted for 1 hour at 40° C. After the reaction, the substrate was washed with pure water.

[0280] (introduction of fluorescent dye) 30 μL of 50 μM Alexa647-C2-Maleimide and 50 μM Maleimido-C3-NTA (5% DMSO 10 mM PB(7.4)) solution was dropped onto the substrate and left to react at 25°C for 1 hour, after which it was washed with pure water. The sample was attached to a flat pipette tip manufactured by Fukae Kasei, and the change in the fluorescence intensity on the surface of the substrate was measured using an automatic pipetting device equipped with a fluorescence microscope. Sequence 1. Chip mounting / Aspirating PBS (150 μL) and acquiring images before antibody immobilization 2. Ni complexation 100 μL of 4 mM NiCl2 (aqueous solution) was aspirated, and the reaction time was 5 min (25°C). Wash 4 times with purified water. Aspirate and dispense 150 μL. 3. proteinG immobilization 1μM His-proteinG (PBS, abcam) aspiration 100μL, reaction 10 min (25℃) Wash 0.01% tween (registered trademark) 20 PBS x2 times, PBS x2 times Aspirate and dispense 150 μL 4. Antibody immobilization 100 nM Anti-CD9 (commercially available) (PBS) 100 μL aspirate, react for 10 min (25°C) Wash 0.01% tween (registered trademark) 20 PBS x2 times, PBS x2 times Aspirate and dispense 150 μL 5. Background (F0: 0.1% BSA, 100 mM PB (pH 7.0), 0.15 M NaCl) measured five times 6. Exosome Adsorption SKBR3 culture supernatant exosomes adjusted to 0, 3, 30, 300, and 1000 fM (0.1% BSA, 100mM PB (pH 7.0), 0.15M NaCl) Aspirate 100 μL of sample (SKBR3-derived exosomes) and react for 5 minutes (25°C). Image acquisition Fluorescence microscope Camera: Zyla4.2 Filter: Cy5 (Excitation wavelength 604-644 nm, Emission wavelength 672-712 nm) Objective lens: x5 Exposure time: 0.5 sec Light output: 12% Light source: mercury lamp Aspirate and dispense 150 μL of 0.01% Tween® 20 PBS Repeat 1 to 6

[0281] (result) For a sensing chip fabricated using anionic polymer-modified silica particles as a cationic surface substrate, a change in relative fluorescence intensity of approximately 5% was confirmed upon addition of 10 fM of exosomes derived from the SK-BR3 cell line.

[0282] Example 10: Biodegradable Nanoparticles: Polylactic Acid / Co-Glycolic Acid (PGLA) [ka]

[0283] (SAM formation on substrate) A gold substrate was immersed in a solution of 0.5 mM 2-(2-bromoisobutyryloxy) undecyl thiol and 0.5 mM Carboxy-EG6 undecane thiol in EtOH to form a SAM (30°C, 20 h).

[0284] (Particle preparation) After ultrasonication (Level 2) of a water suspension of fluorescent carboxylated PGLA nanoparticles (200 nm) for 1 min, the final concentration of nanoparticles and E2 polymer aqueous solution was 2 × 10 11 The solution was mixed to 1 mg / mL with 1.0 × 10 ethanol and diluted 80 times with EtOH. 4 μL of the solution was dropped onto the substrate (1.0 × 10 7 After standing at 25°C for 1 hour, the plate was washed three times with EtOH.

[0285] (SEM observation, fluorescence observation) After mixing the E2 polymer aqueous solution to 1 mg / mL, it was diluted with EtOH and 4 μL was dropped onto the substrate (1.0 × 10 7 particles / 4 μL) (25℃, 1 h) and then washed three times with EtOH. Fluorescence observation of the substrate was performed in EtOH (Keyence BZ-X800, excitation wavelength 470±20 nm / fluorescence wavelength 525±25 nm). After that, it was vacuum dried and gold sputtered, and then a scanning electron microscope (SEM) image was observed (Figure 9).

[0286] (result) The behavior was comparable to that of silica nanoparticles and polystyrene nanoparticles, demonstrating that this method can be applied to any core particle.

[0287] Example 11: Silica Nanoparticles + Cationic Polymer E2P20 Containing Aromatic Moieties 1. E2P20 synthesis method [ka] The cationic polymer E2P20 containing an aromatic moiety (phenyl group) was synthesized as follows. The compounds from the above recipe were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 2 mL). The RAFT agent and initiator were added last. After adding a stirrer, the atmosphere was degassed and replaced with argon, and polymerization was carried out in an oil bath (set temperature: 75 °C) for 24 h (stirring speed: 300 rpm). After the polymerization, the Schlenk flask was cooled with ice and air was introduced to stop the reaction. The reaction solution was added to a large amount of diethyl ether, and the resulting precipitate was collected. The precipitate was washed with diethyl ether and dried in vacuo. 42 μL of the polymer prepared in the previous section, TEA, was dissolved (or dispersed) in CHCl and stirred. 37 mg of N-succinimidyl methacrylate was added and stirred overnight. After the reaction, n-hexane was added to the reaction solution and the resulting precipitate was collected. MeOH, CHCl, and n-hexane were added to the precipitate, and the resulting precipitate was collected and dried in vacuo to obtain the desired polymer (E2P20). Yield: 110 mg [Table E]

[0288] 2. Composite of silica nanoparticles and polymer Silica nanoparticles aqueous dispersion (COOH-terminated, 200 nm, Lot. 0921940-03, 4.0 × 10 11 particles / mL) 40 μL, 40 μL of an aqueous solution of E2P20 (2 mg / mL) was mixed and allowed to stand at 25° C. for 15 minutes to form a complex between the silica nanoparticles and E2P20.

[0289] 3.DLS measurement The silica nanoparticle and E2P20 complex was diluted 100-fold with pure water, and the particle size and Z-potential were measured using DLS.

[0290] 4. Immobilization on the substrate The silica nanoparticle and E2P20 complex was diluted 80-fold with DMF, and 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol:carboxy-EG6undecanethiol (1:1) and left at 25°C for 1 h. The surface was then washed with EtOH and observed by fluorescence microscopy and SEM. The results of particle size measurement by DLS and Z-potential measurement suggested that the cationic polymer was adsorbed onto the silica nanoparticles, changing the surface from a negatively charged state to a positively charged state. Furthermore, the lack of a significant change in particle size indicated that the nanoparticles maintained their dispersibility even after polymer adsorption. [Table F]

[0291] (result) From the observation results shown in Figure 10-1, it was observed that the fluorescent spots derived from silica nanoparticles were monodisperse, not aggregated, and distributed in a monolayer. This indicates that E2P20, in which an aromatic component (phenyl group) has been introduced into E2, can be immobilized to the substrate in a monolayer without being aggregated, when composited with silica nanoparticles, just like E2.

[0292] Example 12: Polystyrene nanoparticles + cationic polymer E2P20 containing aromatic moieties Instead of the silica nanoparticles in Example 11, polystyrene nanoparticles were used to composite the cationic polymer E2P20 containing an aromatic component. [ka] Polystyrene (PS) nanoparticles (250 nm, COOH-terminated, 2.0 × 10 12 20 μL of E2P20 (2 mg / mL) was mixed with 20 μL of an aqueous solution of E2P20 (2 mg / mL), and this solution was diluted 40, 400, 4,000, and 40,000 times with DMF. 4 μL of each solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol and allowed to stand for 1 hour. The surface was then washed with EtOH and observed under a fluorescence microscope (Figure 10-2).

[0293] (result) As with silica nanoparticles, even when polystyrene nanoparticles were used, the fluorescent spots were observed to be monodisperse, non-aggregated, and distributed in a single layer, regardless of the number of particles dropped. This indicates that even when aromatic components are introduced into the cationic polymer, the particles can be fixed in a monodisperse, non-aggregated, and monolayer form.

[0294] (Example 13: Performance test (sensor sensitivity), confirmation of reproducibility) 13-1. Experiment 13-1-1. Surface modification of gold substrate After cleaning the gold substrate (9.8 mm x 4.3 mm) with EtOH, it is treated with UV-O3 for 15 minutes to clean the substrate surface. A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6undecanethiol in a 1:1 ratio in EtOH solvent, and the gold substrate was immersed in this solution. After leaving it at 30 °C for 20 hours, the substrate was washed with EtOH and pure water to form a self-assembled monolayer (SAM) on the gold substrate surface. 13-1-2. Preparation of particle-immobilized substrate Silica nanoparticle dispersion in water (terminal functional group: COOH, particle size 200nm, particle concentration: 4.0×10 11 The complex is formed by mixing the polymer E2 (2.0 mg / mL) with an aqueous solution of polymer E2 (2.0 mg / mL) at a ratio of 1 / 1 (v / v).

[0295] Dilute the above complex solution with DMF and add 1 × 10 8 , 1×10 7 , 1×10 6 , 1×10 5 Add 4 μL of the solution dropwise to the substrate, leaving it to stand at 25°C for 1 hour. Wash the substrate with DMF and then EtOH to obtain the particle-immobilized substrate. Prepare 10 substrates for each addition condition. 13-1-3. Synthesis of polymer matrices and analytical sensors on particle-immobilized substrates A prepolymer solution is prepared with the composition shown in Table 7, and after removing dissolved oxygen by freeze degassing, polymerization is carried out for 20 hours at 25°C in a glove box purged with Ar gas. After polymerization, the substrate is washed with a large amount of pure water.

[0296] After polymerization, the substrate was immersed in AcOH / MeOH (1 / 1, v / v) solution and subjected to ultrasonic irradiation (5 min). The substrate was then washed with MeOH and pure water to remove the silica nanoparticles and form recesses in the polymer thin film. The substrate is then immersed in a 50 mM Tris(2-carboxyethyl)phosphine hydrochloride solution (methanol / pure water = 1 / 1, v / v) and ultrasonicated for 5 minutes to remove the E2 polymer and expose the thiol groups.

[0297] [Table 7]

[0298] 30 μL of 50 μM AlexaFluor® 647 C2 maleimide and 50 μM maleimide C3 NTA PBS solution was dropped onto the substrate and left at 25°C for 1 hour to introduce fluorescent molecules and NTA groups into the recesses. After the reaction, the substrate was washed with MeOH and purified water.

[0299] A 4 mM NiCl2 aqueous solution (30 μL) was dropped onto the aforementioned substrate and allowed to stand at 25°C for 30 minutes to form a Ni-NTA complex. After the reaction, the substrate was washed with pure water. A 1 μM His-tagged Protein G PBS solution (30 μL) was then dropped onto the substrate and allowed to stand at 25°C for 1 hour to introduce Protein G via the formation of a complex between Ni-NTA and the His tag. Finally, a 0.3 μM anti-CD9 antibody PBS solution (30 μL) was dropped onto the substrate and allowed to stand at 25°C for 1 hour to construct the analytical sensor. 13-1.4. Adsorption experiment A fluorescence microscope equipped with an automated pipetting device (System Instruments) was used to conduct exosome capture experiments using prostate cancer cell (PC-3) cells. The PC-3 exosomes were dissolved in PBS (10 mM phosphate, 140 mM NaCl, pH 7.4) at concentrations of 0 and 1.0 fM. The fluorescence microscope measurement conditions were a Cy5 filter (excitation wavelength 604-644 nm, emission wavelength 672-712 nm), a 5x objective lens, a 0.2 sec exposure time, 12% light intensity, and a mercury lamp light source. Measurements were performed within the center of the substrate. The automated pipetting device sequence was as follows: 1. Tip attachment, 2. Sample aspiration (100 μL), 3. Reaction (1 min, 25°C), 4. Total discharge, 5. Aspiration of 150 μL of 10 mM PBS (140 mM NaCl, pH 7.4), and 6. Measurement position retention (repeated from steps 2 to 6). The number of immobilized particles is 1 x 10 8 , 1×10 7 , 1×10 6 , 1×10 5 The initial fluorescence intensity (F0) of 10 analytical sensors fabricated using each substrate and the change in relative fluorescence intensity upon addition of exosomes were measured. The coefficient of variation (= standard deviation / average value [%]) was calculated from the average and standard deviation of the relative fluorescence intensity change of each analytical sensor, and the effect of the number of immobilized particles on reproducibility was examined by examining the correlation between the number of immobilized particles and the coefficient of variation of the fabricated analytical sensors.

[0300] (Comparative Example 1: Silica nanoparticle aggregation and immobilization under the conditions of International Publication No. 2018 / 221271 and Toshifumi Takeuchi et al., J. Am. Chem. Soc., March 10, 2020, Vol. 142, Pages 6617-6624) 1. Introduction of His-tag and thiol groups into silica nanoparticles Fluorescent silica nanoparticles (red-COOH, 200 nm, Lot. 0442240-01) were added at 25 mg / mL (3.0 × 10 12The concentration was adjusted with water to a concentration of 1000 parts per mL. To 1 mL of the silica nanoparticle aqueous suspension, 10 μL of a 50 mM His-tag solution (six histidines attached from the N-terminus, three glycines attached, and a lysine attached to the C-terminus, with the C-terminal carboxyl group amidated) was added, as well as 10 μL of a 50 mM 2-aminoethanthiol-HCl solution and 10 μL of a 50 mM DMT-MM solution. The mixture was reacted at 25°C and 1000 rpm for 19 hours in a thermoshaker to introduce His-tags and thiol groups into the silica nanoparticles. 2. Substrate Preparation A gold substrate is immersed in an EtOH solution containing 0.5 mM 2-(2-bromoisobutyryloxy) undecyl thiol and 0.5 mM Amino-EG6 undecane thiol to introduce amino and bromine groups onto the substrate surface (30°C, 20 h). 3. Introduce NTA onto the substrate and form a nickel complex 80μL of 5mM isothiocyanobenzyl-NTA in DMSO was added dropwise to the substrate with amino and bromine groups and left to stand at 25℃ for 2 hours. The substrate was washed twice with DMSO and twice with pure water and then dried with N2. 30μL of 4mM NiCl2 aqueous solution was added dropwise, left to stand at 25℃ for 15 minutes, and then washed three times with pure water to form a Ni complex of NTA. 4. Immobilization of silica nanoparticles with His-tag and thiol groups 50 μL of silica nanoparticles with His-tags and thiol groups were dropped onto the substrate at the concentrations shown in the table using phosphate-buffered saline (PBS) as the solvent, and allowed to stand at 25°C for 1 hour. The substrate was washed three times with PBS, and the surface of the substrate was observed using a fluorescence microscope (Keyence BZ-800) and SEM (Figure 11). [Table G] The observation results in Figure 11 show that although some individual silica particles are observed, the majority appear to be aggregated. This indicates that when silica nanoparticles are modified with Histag and immobilized on a substrate, they are monodisperse and do not aggregate, and cannot be immobilized into a monolayer.

[0301] (Comparative Example 2: Sensitivity of a sensor made without using particles) [ka] (SAM formation on substrate) A gold substrate (9.8 mm x 4.3 mm) was treated with UV-O3 for 20 minutes to clean the substrate surface. A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyryloxy)undecylthiol and carboxy-EG6 undecanethiol in a 1:1 ratio in EtOH solvent. The gold substrate was then immersed in this solution. After standing at 30°C for 20 hours, the substrate was washed with EtOH to form a self-assembled monolayer (SAM) on the gold substrate surface. An aqueous solution of polymer E2 (1 mg / mL) was diluted 80-fold with DMF, and 4 μL was added dropwise to the SAM-formed substrate and left to stand at 25°C for 1 hour. The substrate was then washed with ethanol.

[0302] (polymer matrix synthesis) A prepolymer solution and an ascorbic acid solution were prepared, and after removing dissolved oxygen by freeze degassing, they were mixed in a glove box and polymerized for 20 hours at 25°C. After polymerization, the substrate was washed with pure water. [Table H]

[0303] (Acetic acid treatment) The substrate was placed in a 50% MeOH, 50% acetic acid solution, subjected to ultrasonic treatment for 5 minutes, and then washed with pure water.

[0304] (SS reductive cleavage) A 0.5 mM aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride was placed in a thermoshaker and preheated to 40°C for 5 minutes, after which the acetic acid-treated substrate was placed therein and reacted for 1 minute at 40°C. After the reaction, the substrate was washed with pure water.

[0305] (fluorescence, NTA introduction) 30 μL of a solution of 50 μM maleimido-C3-NTA and 50 μM Alexa 647-C2-maleimide in 10 mM PBB (pH 7.4) containing 20% ​​DMSO was added dropwise, and the mixture was left to react at 25°C for 1 hour, after which it was washed with DMSO and pure water.

[0306] (His-tagged Protein G, antibody introduction) 30 μL of a 4 mM NiCl2 solution was dropped onto the substrate and allowed to stand (25°C, 15 min). After the reaction, the substrate was washed with pure water. 30 μL of a 1 μM His-tagged protein G (His-Protein G) solution in PBS was dropped onto the substrate and allowed to stand at 25°C for 1 hour to immobilize His-Protein G. After His-Protein G immobilization, the substrate was washed with PBS. The antibody was introduced into the substrate by dropping 30 μL of a 0.1 μM anti-CD9 solution in PBS and leaving it to stand for 1 hour at 25° C. After the antibody introduction, the substrate was washed with PBS. The sample was attached to a flat pipette tip manufactured by Fukae Kasei, and the change in the fluorescence intensity on the surface of the substrate was measured using an automatic pipetting device equipped with a fluorescence microscope. The concentrations of PC3 exosome particles in PBS solution were 0.03, 0.3, 3, and 30 fM. 1. Tip installation 2. Aspirate 150 μL of PBS and perform 5 F0 measurements. 3. Aspirate 100 μL of sample (PC3-derived exosomes) 4. Reaction: 5 min (25°C) 5. Full discharge 6. Aspirate 150μL of 10mM PBS (140mM NaCl, pH7.4) 7. Automatic Measurement Fluorescence microscope Camera: Zyla4.2 Filter: Cy5 (Excitation wavelength 604-644 nm, Emission wavelength 672-712 nm) Objective lens: x5 Exposure time: 0.5 sec Light output: 12% Light source: mercury lamp 8. Repeat steps 3 to 7

[0307] (result) The results are shown in Figure 12. In the non-porous substrate without a silica particle core, there was no response to exosomes.

[0308] Example 14: Animal Experiment In this example, a demonstrative example based on an animal experiment is shown. Exosomes in the blood of dogs and cats are measured. Blood samples are taken from dogs and cats with cancer or visceral disease that has been confirmed by a veterinarian, and exosomes are measured. After the animals have fully recovered, exosomes are measured again, and by observing the differences, it can be considered whether cancer or visceral disease in dogs and cats can be detected.

[0309] Other possible uses include: Non-human use 1. Testing pets (cats, dogs, birds, etc.) for diseases including cancer 2. Infectious diseases caused by viruses and infectious diseases carried by livestock (cattle, horses, pigs, chickens, etc.) Detection of proteins (e.g., prions) and other pathogenic / infectious agents 3. Infectious diseases carried by harmful animals (birds, bats, foxes, mongooses, raccoons, etc.) Detection of causative viruses, infectious proteins (e.g., prions), and other pathogenic / infectious agents 4. Checking the quality and freshness of seafood 5. Checking the quality and freshness of agricultural products 6. Check for pathogens and viruses in plants and trees Example 15: Clinical application In this example, the application in cancer diagnosis is demonstrated as follows.

[0310] In clinical research approved by the Clinical Research Review Board of the hospital where the clinical experiment was conducted, and with the patient's consent, we will examine whether this sensor can detect cancer by observing differences in sensor response before and after total resection surgery. We will also examine whether it is possible to check for cancer metastasis and recurrence through continuous observation. We will examine whether it is possible to evaluate the effectiveness of drug therapy by looking at differences in sensor response before and after medication. We will examine whether lifestyle-related diseases can be diagnosed from the relationship between ingested food and sensor response. We will also examine whether the presence of cancer cell surface antigens can be estimated from sensor response and whether companion diagnostics are possible. Furthermore, we will examine whether it is possible to use sensor response to control the quality of cells in cell-based therapies.

[0311] In addition to the above, the technology of the present disclosure is expected to have the following applications. Cancer testing Checking for residual tissue during cancer surgery Checking for cancer metastasis and recurrence Therapeutic effects of drug therapy Lifestyle-related disease testing Companion diagnostics for medication Quality control of cells in cell engineering (Note) While the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present disclosure should be construed solely in terms of the claims that follow. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0312] The techniques provided in this disclosure can be used in any field (including diagnostics) that utilizes laboratory analytical techniques.

Claims

1. A) a substrate body; B) a polymer matrix disposed on the substrate body, the polymer matrix having a recess that at least partially fits a detection target; C) a signal substance binding group arranged in the recess; D) a binding group for a specific binding molecule that binds to a molecule to be detected, the specific binding molecule being disposed in the recess, wherein the recess contains 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 The analytical sensors are arranged on the substrate body at a density of 1000 nm to 1000 nm without clumping.

2. The recess is 1×10 4 pieces / mm 2 ~1×10 6 pieces / mm 2 2. The analytical sensor of claim 1, wherein the sensor is present without aggregation at a density of 0.1 to 1.

0.

3. 1. A method for producing a substrate for preparing an analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate body so that the particles are disposed on the substrate body without agglomeration; C) providing a polymer matrix raw material to the substrate body having the particles fixed thereto; D) forming a substrate body having the polymer matrix disposed thereon by subjecting the substrate body to conditions under which the polymer matrix polymerizes; E) forming recesses by subjecting the substrate to conditions that dissociate the particles from the substrate body; encompassing, method.

4. 1. A method for manufacturing an analytical sensor, comprising: A) providing particles; B) adding the particles to a substrate such that the particles are disposed on the substrate without agglomeration; C) providing a polymer matrix material to the substrate having the particles fixed thereto; D) forming a substrate body having the polymer matrix disposed thereon by subjecting the substrate body to conditions under which the polymer matrix polymerizes; E) subjecting the substrate body to conditions that dissociate the particles from the substrate body, thereby forming recesses; F) binding a substance required for analysis to the recess. encompassing, method.

5. 1×10 10 pieces / mm 2 5. A method for manufacturing an analytical sensor as described in claim 4, wherein by forming recesses at a density below 20%, 80% or more of the analytical sensors are produced having a coefficient of variation of relative fluorescence intensity change in target substance detection of 20% or less.

6. 5. The method for manufacturing the analytical sensor according to claim 4, wherein the analytical sensors having a coefficient of variation of relative fluorescence intensity change in target substance detection of 20% or less are produced at a rate of 80% or more by forming recesses on the substrate whose area occupancy is equal to or less than closest packing.

7. The step of adding particles so as to be arranged without agglomeration may involve adding particles to the substrate in an amount of 1.0×10 0 particles / μL to 1.0 x 10 10 at a concentration of 1.0 x 10 per μL or per surface area of ​​substrate 0 pieces / mm 2 to 1.0×10 10 pieces / mm 2 7. The method of claim 3, further comprising adding an amount of

8. The step of disposing without aggregation may comprise disposing 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 7. The method of claim 3, further comprising the step of disposing the particles at a density of

9. The method according to any one of claims 3 to 6, wherein the particles are in a single layer state.

10. The method of claim 8, wherein the particles are in a single layer.

11. In the step of forming the recess, the recess is formed to have a thickness of 1×10 3 pieces / mm 2 ~1×10 10 pieces / mm 2 The method of any one of claims 3 to 6, wherein the granules are present at a density of

12. The method according to any one of claims 3 to 6, wherein the step of disposing the particles comprises a spin coating method, a method of dropping particles onto a substrate, a method of immersing a substrate in a particle dispersion, a method of lifting a substrate from a particle dispersion, a method of spraying a particle dispersion, or a printing method.

13. 8. The method according to claim 7, wherein the step of disposing the particles comprises a spin coating method, a method of dropping particles onto a substrate, a method of immersing a substrate in a particle dispersion, a method of lifting a substrate from a particle dispersion, a method of spraying a particle dispersion, or a printing method.

14. 9. The method according to claim 8, wherein the step of disposing the particles comprises a spin coating method, a method of dropping particles onto a substrate, a method of immersing a substrate in a particle dispersion, a method of lifting a substrate from a particle dispersion, a method of spraying a particle dispersion, or a printing method.

15. 10. The method according to claim 9, wherein the step of disposing the particles comprises a spin coating method, a method of dropping particles onto a substrate, a method of immersing a substrate in a particle dispersion, a method of lifting a substrate from a particle dispersion, a method of spraying a particle dispersion, or a printing method.

16. 11. The method according to claim 10, wherein the step of disposing the particles comprises a spin coating method, a method of dropping particles onto a substrate, a method of immersing a substrate in a particle dispersion, a method of lifting a substrate from a particle dispersion, a method of spraying a particle dispersion, or a printing method.

17. 12. The method according to claim 11, wherein the step of disposing the particles comprises a spin coating method, a method of dropping particles onto a substrate, a method of immersing a substrate in a particle dispersion, a method of lifting a substrate from a particle dispersion, a method of spraying a particle dispersion, or a printing method.

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