Technique for aligning particles on substrate without agglomeration

Abstract

Provided is a novel technique for providing a base material that is useful when an analysis sensor is produced. A base material for producing an analysis sensor provided by the present disclosure has specific characteristics (e.g., is a monodispersion or a monolayer, or has a specific density), thereby making it possible to produce an analysis sensor that is stable and enables high-accuracy analysis. In the present disclosure, it is not necessary to eliminate a surplus due to fine particles being overabundantly arranged on a base material, repeatability of the density of the fine particles anchored to the base material is improved, and monodispersion properties and monolayer properties are also improved.

Description

[Technical Field]

[0001] This invention relates to a technique for arranging particles on a substrate without agglomeration, and its application. More specifically, this invention relates to a substrate for fabricating an analytical sensor for a test object configured on a support, a method for manufacturing the same, an analytical sensor for the test object, a method for manufacturing the same, and a method for analyzing the test object. [Previous Technology]

[0002] Small extracellular vesicles (sEVs) are one of the endoplasmic reticulum vesicles released from cells, and are lipid double-membrane vesicles with a diameter of 20–200 nm. Small extracellular vesicles contain proteins and nucleic acids such as miRNA and mRNA, and also have proteins on their surface. Because small extracellular vesicles are characterized by these substances, it is believed that by analyzing the characteristics of small extracellular vesicles, it is possible to infer the type of cell that secretes them. Furthermore, small extracellular vesicles have been confirmed to exist in various body fluids, making them relatively easy to collect.

[0003] Small extracellular vesicles secreted by cancer cells contain substances from the tumor. Therefore, it is hoped that cancer can be diagnosed by analyzing the substances contained in small extracellular vesicles in body fluids. Furthermore, since small extracellular vesicles are actively secreted by cells, it is predicted that certain characteristics will be present even in the early stages of cancer. [Summary of the Invention]

[0004] [Technical means to solve the problem]

[0005] This invention provides a novel technique for providing an advantageous substrate when fabricating analytical sensors. Further, the analytical sensor fabrication substrate provided by this invention, by having specific characteristics (e.g., monodisperse, monolayer, or specific density), can fabricate stable analytical sensors capable of high-precision analysis. This invention improves the reproducibility of the density of microparticles immobilized on the substrate by eliminating the need to place excessive microparticles on the substrate and then remove the excess, and also improves monodispersity and monolayer properties. In a preferred embodiment of this invention, monodisperse microparticle-immobilized substrates and / or monolayer microparticle-immobilized substrates can be reproducibly obtained by immobilization using the electrostatic interaction between the substrate and the particles, and by dispersing and / or layering the particles on the substrate.

[0006] The present invention provides, for example, the following items.

[0007] (Item A1) A substrate for fabricating an analytical sensor, comprising A) a substrate body and B) particles monodispersed in the substrate body. (Item A2-1) A substrate for fabricating an analytical sensor, comprising A) a substrate body and B) particles disposed in the substrate body at a density of 1×10³ particles / mm² to 1×10⁸ particles / mm². (Item A2-2) A substrate for fabricating an analytical sensor, comprising A) a substrate body and B) particles disposed in the substrate body at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm². (Item A2-3) The substrate for fabricating an analytical sensor as described in any of the preceding items, wherein the particles comprise a substance used in the analytical sensor. (Item A3) The substrate for fabricating an analytical sensor as described in any of the preceding items, wherein the particles comprise particles integrated with a modifying substance. (Item A4) A substrate for fabricating an analytical sensor as described in any of the above items, wherein the substrate further comprises C) a polymer matrix disposed on the substrate body, and the polymer matrix having at least a portion of recesses for adaptors; the particles are disposed in the recesses. (Item B1A) A convex analytical sensor comprising: A) a substrate body; B) a polymer matrix disposed on the substrate body, and the polymer matrix having at least a portion of recesses for adaptors, the particles being disposed in the recesses; C) a binding base for a signaling substance disposed on the particles; and D) a binding base for a specific binding molecule disposed on the particles to bind with a molecule that is a detection target; wherein the particles are disposed on the substrate body at a density of 1×10³ particles / mm² to 1×10⁸ particles / mm². (Item B1B) A convex analysis sensor comprising: A) a substrate body; B) a polymer matrix disposed on the substrate body, the polymer matrix having at least a portion of a recess for an adaptee, the particle being disposed in the recess; C) a binding base for a signaling substance disposed on the particle; and D) a binding base for a specific binding molecule disposed on the particle to bind with a molecule that is a detection target; wherein the particle portion is disposed on the substrate body at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm². (Item B1C) An analytical sensor comprising: A) a substrate body; B) a polymer matrix disposed on the substrate body, wherein the polymer matrix has at least a portion of recesses for adapting target molecules; C) a binding base for a signal substance disposed in the recesses; and D) a binding base for a specific binding molecule disposed in the recesses for binding to a molecule that is a target molecule; wherein the recesses are disposed on the substrate body at a density of 1×10³ / mm² to 1×10⁸ / mm².(Item B1D) An analytical sensor comprising: A) a substrate body; B) a polymer matrix disposed on the substrate body, wherein the polymer matrix has at least a portion of recesses for adapting target molecules; C) a binding base for a signal substance disposed in the recesses; and D) a binding base for a specific binding molecule disposed in the recesses for binding to a molecule that is a target molecule; wherein the recesses are disposed on the substrate body at a density of 1×10³ / mm² to 1×10¹⁰ / mm². (Item B2) An analytical sensor as described in any of the preceding items, wherein the recesses are present at a density of 1×10⁴ / mm² to 1×10⁶ / mm². (Item C1A) A method for manufacturing a substrate for a convex analytical sensor, comprising: A) a step of providing particles; B) a step of adding the particles to a substrate in a monodisperse manner on the substrate body; C) a step of providing a raw material for a polymer matrix to the substrate on which the particles are fixed; and D) a step of forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to polymerization conditions on the polymer matrix. (Item C1) A method for manufacturing a substrate for a analytical sensor, comprising: A) a step of providing particles; B) a step of adding the particles to a substrate in a monodisperse manner on the substrate body; C) a step of providing a raw material for a polymer matrix to the substrate on which the particles are fixed; D) a step of forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to polymerization conditions on the polymer matrix; and E) a step of forming a recess by subjecting the particles to dissociation from the substrate. (Item C1-1) The method described in any of the above items, wherein the step of adding the particles in a monodisperse configuration includes adding particles at a concentration of 1.0 × 10⁰ particles / μL to 2.5 × 10⁸ particles / μL per square mm of substrate, or at an amount of 1.0 × 10⁰ particles to 2.5 × 10⁸ particles. (Item C1-2) The method described in any of the above items, wherein the step of adding the particles in a monodisperse configuration includes adding particles at a concentration of 1.0 × 10⁰ particles / μL to 1.0 × 10¹⁰ particles / μL per square mm of substrate, or at an amount of 1.0 × 10⁰ particles to 1.0 × 10¹⁰ particles. (Item C2A) A method for manufacturing a sensor for convex analysis, comprising: A) a step of providing particles; B) a step of adding the particles to a substrate in such a way that the particles are monodispersed in the substrate body; C) a step of providing a raw material for a polymer matrix to the substrate on which the particles are fixed; D) a step of forming a substrate having the polymer matrix disposed thereon by providing the substrate with conditions for polymerization of the polymer matrix; and F) a step of binding the analytical material to the particles.(Item C2) A method for manufacturing an analytical sensor, comprising: A) a step of providing particles; B) a step of adding the particles to a substrate in a monodisperse manner on the substrate body; C) a step of providing a polymer matrix raw material to the substrate on which the particles are fixed; D) a step of forming a substrate having the polymer matrix disposed thereon by providing the substrate with conditions for polymerization of the polymer matrix; E) a step of forming recesses by providing conditions for dissociation of the particles from the substrate; and F) a step of binding the analytical material to the recesses. (Item C3A-1) The method for manufacturing an analytical sensor as described in any of the above items, wherein the analytical sensor is manufactured with a coefficient of variation of 20% or less at a rate of 80% or more by forming recesses with a density of 2.0 × 10⁷ particles / mm² or less. (Item C3A-2) A method for manufacturing an analytical sensor as described in any of the above items, wherein the analytical sensor with a coefficient of variation of 20% or less is produced at a ratio of 80% or more by forming recesses with a density of 1.0 × 10¹⁰ particles / mm² or less. (Item C3B) A method for manufacturing an analytical sensor as described in any of the above items, wherein the analytical sensor with a coefficient of variation of 20% or less is produced at a ratio of 80% or more by forming recesses on a substrate with an area occupancy of at least dense filling. (Item C4A) A method as described in any of the above items, wherein the step of configuring the particles monodisperse includes the step of configuring the particles on a substrate at a density of 1 × 10³ particles / mm² to 1 × 10⁸ particles / mm². (Item C4B) The method described in any of the above items, wherein the step of configuring the particles monodisperse includes the step of configuring the particles on the substrate at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm². (Item C5A) The method described in any of the above items, wherein in the step of forming the recess, the monodisperse recess exists at a density of 1×10³ particles / mm² to 1×10⁸ particles / mm². (Item C5B) The method described in any of the above items, wherein in the step of forming the recess, the monodisperse recess exists at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm². (Item C6) The method described in any of the above items, wherein the step of configuring the particles includes spin coating, dropping particles onto a substrate, immersing the substrate in a particle dispersion, lifting the substrate from the particle dispersion, or spraying the particle dispersion. (Item 1) A substrate for fabricating an analytical sensor, comprising A) a substrate body and B) particles disposed on the substrate body in a single layer state.(Item 2) A substrate for fabricating an analytical sensor as described in any of the preceding items, wherein the particles are disposed without aggregation. (Item 1A) A substrate for fabricating an analytical sensor, comprising A) a substrate body and B) particles disposed on the substrate body without aggregation. (Item 2A) A substrate for fabricating an analytical sensor as described in any of the preceding items, wherein the particles are in a single-layer state. (Item 3) A substrate for fabricating an analytical sensor as described in any of the preceding items, wherein the particles include particles integrated with a modifying substance. (Item 4) A substrate for fabricating an analytical sensor as described in any of the preceding items, wherein the substrate further comprises C) a polymer matrix disposed on the substrate body, and the polymer matrix has at least a portion, preferably adapted to the object for functioning as an analytical sensor, wherein the particles are disposed in the recess. (Item 5) A convex analysis sensor comprising: A) a substrate body; B) a polymer matrix disposed on the substrate body, wherein the polymer matrix has at least a portion of a recess adapted to a detection target, the particles being disposed in the recess without agglomeration; C) a binding base for a signaling substance disposed on the particles; and D) a binding base for a specific binding molecule disposed on the particles to bind with a molecule that is a detection target; wherein the particles are disposed on the substrate body at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm². (Item 6) An analytical sensor comprising: A) a substrate body; B) a polymer matrix disposed on the substrate body, wherein the polymer matrix has at least a portion of recesses adapted to a detection target; C) a binding base for a signal substance disposed in the recesses; and D) a binding base for a specific binding molecule disposed in the recesses to bind to a molecule that is a detection target; wherein the recesses are disposed on the substrate body at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm² without aggregation. (Item 7) An analytical sensor as described in any of the preceding items, wherein the aforementioned recesses are present at a density of 1×10⁴ particles / mm² to 1×10⁶ particles / mm² without aggregation. (Item 8) A method for manufacturing a substrate for a convex analysis sensor, comprising: A) a step of providing particles; B) a step of adding the particles to the substrate in such a way that the particles are disposed on the substrate body without agglomeration; C) a step of providing the substrate with the particles fixed with a raw material of a polymer matrix; and D) a step of forming a substrate with the polymer matrix disposed thereon by providing the substrate with conditions for polymerization of the polymer matrix.(Item 9) A method for manufacturing a substrate for an analytical sensor, comprising: A) providing particles; B) adding the particles to the substrate in a manner that prevents the particles from agglomerating and is disposed on the substrate body; C) providing the substrate on which the particles are fixed with a raw material of a polymer matrix; D) forming a substrate disposed with the polymer matrix by providing the substrate with conditions for polymerization of the polymer matrix; and E) forming recesses by providing conditions for the particles to dissociate from the substrate. (Item 10) The method as described in any of the preceding items, wherein the step of adding the particles in a manner that prevents agglomeration comprises adding the particles to the substrate at a concentration of 1.0 × 10⁰ particles / μL to 1.0 × 10¹⁰ particles / μL, or at an amount of 1.0 × 10⁰ particles / mm² to 1.0 × 10¹⁰ particles / mm² relative to the surface area of ​​the substrate. (Item 11) A method for manufacturing a convex analytical sensor, comprising: A) a step of providing particles; B) a step of adding the particles to a substrate in a manner that prevents the particles from agglomerating and disposing them on the substrate body; C) a step of providing a raw material for a polymer matrix to the substrate on which the particles are fixed; D) a step of forming a substrate on which the polymer matrix is ​​disposed by subjecting the substrate to polymerization conditions; and E) a step of binding an analytical substance to the particles. (Item 12) A method for manufacturing an analytical sensor, comprising: A) a step of providing particles; B) a step of adding the particles to a substrate in a manner that prevents the particles from agglomerating and disposing them on the substrate body; C) a step of providing a raw material for a polymer matrix to the substrate on which the particles are fixed; D) a step of forming a substrate on which the polymer matrix is ​​disposed by subjecting the substrate to polymerization conditions; E) a step of forming a recess by subjecting the particles to dissociation from the substrate; and F) a step of binding an analytical substance to the recess. (Item 13) The method for manufacturing an analytical sensor as described in any of the preceding items, wherein the analytical sensor is manufactured at a ratio of 80% or more by forming recesses with a density of 1.0 × 10¹⁰ particles / mm² or less, and the coefficient of variation of the relative fluorescence intensity change in target material detection is 20% or less. (Item 14) The method for manufacturing an analytical sensor as described in any of the preceding items, wherein the analytical sensor is manufactured at a ratio of 80% or more by forming recesses on a substrate with an area occupancy of at least dense filling, and the coefficient of variation of the relative fluorescence intensity change in target material detection is 20% or less. (Item 15) The method as described in any of the preceding items, wherein the step of non-agglomerated arrangement includes the step of arranging the particles on a substrate at a density of 1 × 10³ particles / mm² to 1 × 10¹⁰ particles / mm².(Item 16) The method as described in any of the preceding items, wherein the particles are in a single layer state. (Item 17) The method as described in any of the preceding items, wherein in the step of forming the recess, the recess exists at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm². (Item 18) The method as described in any of the preceding items, wherein the step of configuring the particles includes spin coating, dropping particles onto a substrate, immersing a substrate in a particle dispersion, lifting the substrate from the particle dispersion, or spraying the particle dispersion.

[0008] In this invention, it is intended that one or more of the above-described features can be further combined in addition to the explicitly stated combinations. Further embodiments and advantages of this invention can be understood by reading the following detailed description as needed. [Effects of the Invention]

[0009] By utilizing the related technology of the present invention, in the technique of creating pores of target molecules using particles through molecular imprinting, it is possible to form monodisperse or monolayer pores on a substrate, thereby improving the reproducibility of pore formation. The method of the present invention occupies an important position in the fundamental technology for fabricating uniform sensor substrates.

Implementation Method

[0011] Hereinafter, the present invention will be described in more detail.

[0012] Throughout this invention, it should be understood that singular expressions also include the concept of their plural forms, unless otherwise specifically mentioned. Therefore, it should be understood that singular expressions (e.g., "a," "an," "the," etc. in English) also include the concept of their plural forms, unless otherwise specifically mentioned. Furthermore, it should be understood in this specification that the terms used are used in their commonly understood meanings in the art, unless otherwise specifically mentioned. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In the event of any conflict, this invention (including the definitions) takes precedence.

[0013] (Definitions) First, the terms and general techniques used in this specification will be explained.

[0014] In this specification, "substrate" refers to the material that forms the basis of the analytical sensor. The substrate material may be, for example, materials selected from the group consisting of metals, metal oxides, glass, paper (cellulose), cloth, silicon dioxide, silicon, and resins, and combinations thereof. Examples of metals include gold, silver, copper, aluminum, titanium, tungsten, and molybdenum, but are not limited to these. Examples of resins include poly(meth)acrylate, polystyrene, ABS (acrylonitrile-butadiene-styrene copolymer), polycarbonate, polyester, polyethylene, polypropylene, nylon, polyurethane, silicone resin, fluoropolymer, methylpentene resin, phenolic resin, melamine resin, epoxy resin, and vinyl chloride resin, but are not limited to these.

[0015] In this specification, "monodisperse" means that the dispersed phase in a dispersion system is of uniform size. When describing a substrate such as a substrate for manufacturing an analytical sensor, "monodisperse" means that the particles are substantially uniformly disposed on the substrate. In the case of a monodisperse configuration, it is advantageous to be disposed without agglomeration. The density used to achieve monodispersity can vary depending on the intended target of the final analytical sensor, preferably about 10³ particles / mm² to about 10⁸ particles / mm², more preferably about 10⁴ particles / mm² to about 10⁷ particles / mm², and even more preferably about 10⁴ particles / mm² to about 10⁶ particles / mm². As upper limits for density, examples include approximately 1×10⁷ particles / mm², approximately 9×10⁶ particles / mm², approximately 8×10⁶ particles / mm², approximately 7×10⁶ particles / mm², approximately 6×10⁶ particles / mm², approximately 5×10⁶ particles / mm², approximately 4×10⁶ particles / mm², approximately 3×10⁶ particles / mm², approximately 2×10⁶ particles / mm², and approximately 1×10⁶ particles / mm². As lower limits, examples include approximately 1×10³ particles / mm², approximately 2×10³ particles / mm², approximately 3×10³ particles / mm², approximately 4×10³ particles / mm², approximately 5×10³ particles / mm², approximately 6×10³ particles / mm², approximately 7×10³ particles / mm², and approximately 8×10⁶ particles / mm². 3 per mm², approximately 9 × 10³ per mm², approximately 1 × approximately 10⁴ per mm², etc., or preferably approximately 10³ per mm² to approximately 10¹⁰ per mm², more preferably approximately 10⁴ per mm² to approximately 10⁹ per mm², and even more preferably approximately 10⁴ per mm² to approximately 10⁸ per mm². As upper limits for density, examples include approximately 1×10¹⁰ particles / mm², approximately 9×10⁹ particles / mm², approximately 8×10⁹ particles / mm², approximately 7×10⁹ particles / mm², approximately 6×10⁹ particles / mm², approximately 5×10⁹ particles / mm², approximately 4×10⁹ particles / mm², approximately 3×10⁹ particles / mm², approximately 2×10⁹ particles / mm², and approximately 1×10⁹ particles / mm². As lower limits, examples include approximately 1×10³ particles / mm², approximately 2×10³ particles / mm², approximately 3×10³ particles / mm², approximately 4×10³ particles / mm², approximately 5×10³ particles / mm², approximately 6×10³ particles / mm², approximately 7×10³ particles / mm², and approximately 8×10⁹ particles / mm². 3 pieces / mm², approximately 9×10³ pieces / mm², approximately 1×approximately 10⁴ pieces / mm², etc.

[0016] In this specification, "monolayer" means a layer of particles that do not overlap. When configured in a monolayer, it is advantageous to configure without agglomeration. The density used to achieve the monolayer can be varied depending on the intended target of the final analytical sensor, preferably about 10³ particles / mm² to about 10⁸ particles / mm², more preferably about 10⁴ particles / mm² to about 10⁷ particles / mm², and even more preferably about 10⁴ particles / mm² to about 10⁶ particles / mm². As upper limits for density, examples include approximately 1×10⁷ particles / mm², approximately 9×10⁶ particles / mm², approximately 8×10⁶ particles / mm², approximately 7×10⁶ particles / mm², approximately 6×10⁶ particles / mm², approximately 5×10⁶ particles / mm², approximately 4×10⁶ particles / mm², approximately 3×10⁶ particles / mm², approximately 2×10⁶ particles / mm², and approximately 1×10⁶ particles / mm². As lower limits, examples include approximately 1×10³ particles / mm², approximately 2×10³ particles / mm², approximately 3×10³ particles / mm², approximately 4×10³ particles / mm², approximately 5×10³ particles / mm², approximately 6×10³ particles / mm², approximately 7×10³ particles / mm², and approximately 8×10⁶ particles / mm². 3 per mm², approximately 9 × 10³ per mm², approximately 1 × approximately 10⁴ per mm², etc., or preferably approximately 10³ per mm² to approximately 10¹⁰ per mm², more preferably approximately 10⁴ per mm² to approximately 10⁹ per mm², and even more preferably approximately 10⁴ per mm² to approximately 10⁸ per mm². As upper limits for density, examples include approximately 1×10¹⁰ particles / mm², approximately 9×10⁹ particles / mm², approximately 8×10⁹ particles / mm², approximately 7×10⁹ particles / mm², approximately 6×10⁹ particles / mm², approximately 5×10⁹ particles / mm², approximately 4×10⁹ particles / mm², approximately 3×10⁹ particles / mm², approximately 2×10⁹ particles / mm², and approximately 1×10⁹ particles / mm². As lower limits, examples include approximately 1×10³ particles / mm², approximately 2×10³ particles / mm², approximately 3×10³ particles / mm², approximately 4×10³ particles / mm², approximately 5×10³ particles / mm², approximately 6×10³ particles / mm², approximately 7×10³ particles / mm², and approximately 8×10⁹ particles / mm². 3 pieces / mm², approximately 9×10³ pieces / mm², approximately 1×approximately 10⁴ pieces / mm², etc.

[0017] In this specification, the term "agglomeration," when used to describe particles, refers to the aggregation of multiple particles through mutual attraction. "Non-existence," "agglomeration," or "non-agglomeration" refers to the presence of particles in a state where this aggregation state is substantially non-existent (i.e., below the detection limit). Regarding non-agglomeration, it typically refers to the absence of particles that are substantially non-existent in any overlapping and fixed portion during SEM measurement, preferably less than 10% of the total, more preferably less than 5%, 4%, 3%, 2%, or 1%. The SEM measurement method is as follows: the object to be measured is vacuum dried and then gold sputtered, with conditions set to achieve a magnification of 2000x or higher. In cases where SEM cannot provide a judgment, observation can be performed using an atomic force microscope. Using the technology of this invention, particles can be disposed on a substrate without agglomeration.

[0018] In this specification, "Single Layer" typically refers to a portion that is substantially absent from the three-dimensional overlap and fixation during SEM measurement, preferably less than 10% of the total, more preferably less than 5%, 4%, 3%, 2%, or 1%. Whether it is a single layer is preferably determined by taking multiple locations as objects, preferably n=5, more preferably n=10 or more locations as measurement objects, and calculating their average value. The determination of a single layer can also be made by vacuum drying the measurement object and then performing gold sputtering to achieve a magnification of 2000x or higher. Regarding the determination of a single layer, when it is not possible to make a determination using SEM, it can also be observed using an atomic force microscope. If the technology of this invention is used, particles can be disposed on a substrate as a single layer.

[0019] In this specification, "particle" refers to a basic substance having a structure that serves as a template for forming a recess in a substrate in an analytical sensor. This substance may contain biological molecules or a particle nucleus (e.g., a substance containing a particle nucleus and a modifying substance). Each particle typically contains the substance used in the analytical sensor. The particle nucleus is not particularly limited as long as it can be used as a template in molecular imprinting, including artificially manufactured inorganic and organic particles. When the size is in the nm range, it is sometimes referred to as a particle nucleus, but this is not strictly distinguished in this invention. When referred to as a particle nucleus, unless specifically limited in size, it is understood to include those with larger or smaller nuclei. Examples of inorganic particles include: metals, metal oxides, nitrides, fluorides, sulfides, borides, and their complexes, as well as hydroxyapatite, with silicon dioxide (silicon oxide) being a preferred example. Furthermore, examples of organic particles include: latex curing agents, dextran, polyglucosamine, polylactic acid, poly(meth)acrylic acid, polystyrene, polyethylimide, etc., but are not limited to these. Also, particles with binding functional groups attached to the surface of biological molecules or particle nuclei can be used. The particle size can vary depending on the intended target of the final analytical sensor, and examples include approximately 1 nm to approximately 100 μm, approximately 1 nm to approximately 20 nm, approximately 20 nm to approximately 500 nm, approximately 50 nm to approximately 200 nm, approximately 100 nm to approximately 500 nm, approximately 1 μm to approximately 10 μm, approximately 10 μm to approximately 100 μm, etc., but are not limited to these. The particles used in this invention can also be particles formed by integrating a modifying substance with a particle nucleus.

[0020] In this specification, a "modifying substance" is a substance that has the purpose of directly or indirectly imparting a desired function to an analytical sensor or a substrate for manufacturing an analytical sensor. To achieve this imparting of function, it may contain various functional groups, or form a structure in which various functional groups may be contained. In this invention, a modifying substance is advantageously a substance capable of interacting with a particle nucleus, preferably a substance that can be integrated with a particle nucleus by utilizing interactions that can be separated without damaging the substrate, and / or a substance that can be integrated with a particle nucleus by utilizing interactions that can be separated without damaging the particle nucleus. The following substances are representative examples of modifying substances. More specifically, examples include polyisopropylacrylamide copolymers, poly(meth)acrylamide copolymers, polylactic acid derivatives, poly(meth)acrylic acid copolymers, poly(meth)acrylate copolymers, poly(meth)acrylate hydroxyethyl ester copolymers, glucosamine derivatives, and polylysine derivatives, as well as combinations thereof, which are detailed elsewhere in this specification, but are not limited to these. Furthermore, one or more modifying substances can also be integrated with the particle nucleus.

[0021] In this specification, "integration" generally refers to substances becoming a single entity. When describing modified substances and particle nuclei, integration can be achieved by any action as long as the state during the manufacture of the analytical sensor or the substrate for manufacturing the analytical sensor is such that it will only disintegrate under conditions that allow it to be separated without damaging the particle nucleus and / or without damaging the substrate. Examples of such actions include covalent bonds and non-covalent bonds.

[0022] In this specification, "substances used in analytical sensors" refers to substances used directly or indirectly in the analytical sensor or its manufacturing process. Examples include substances with binding bases for molecules that specifically bind to target substances, substances with reversible binding bases, and binding bases for signal substances, but are not limited to these.

[0023] In this specification, "substrate for fabricating an analytical sensor" refers to any substrate used to fabricate an analytical sensor, and any substrate can be used as long as its shape and material are suitable for the analytical sensor. Preferably, the substrate for fabricating an analytical sensor is a substrate with recesses. Here, it is advantageous that by modifying the recesses with binding groups for specifically binding molecules and binding groups for signal substances, the user can easily customize the sensor to detect the target object with higher sensitivity. In this specification, "substrate for fabricating an analytical sensor" is also referred to as "measuring substrate".

[0024] In this specification, "density" refers to the number of particles present per unit area. For example, particles / mm² refers to the number of particles present per 1 mm². The method for determining density is arbitrary. For example, density can be determined by using the particle count function of the analysis software provided with the measuring device or free image analysis software such as ImageJ (https: / / imagej.nih.gov / ij / ) based on the results obtained after observing the surface using a fluorescence microscope or an electron microscope.

[0025] In this specification, "modified material integrated particle" refers to a particle formed by integrating a modifying material and a particle nucleus, and refers to a particle formed by integrating a material and a particle having a base for disposing of a binding base in a molecular imprinting recess.

[0026] In this specification, "base" refers to a monovalent base unless otherwise specified. Examples of non-monovalent bases include alkyl groups (divalent). Also, the term "base" is sometimes omitted when used in this specification.

[0027] In this specification, "reversible linking base" refers to a base that cuts (breaks) and forms direct bonds or bases with two or more valences that are reversible. Specific examples may be the bases shown in Table 1A 1-3 below, but are not limited to these.

[0028] [Table 1A-1] 1-1 1-2 1-3 1-4 Binding functional groups Corresponding binding functional groups Reversible linker Reversible key style (A binding group for molecules that specifically bind to the target molecule) (binding group) (Reversible linker) (Binding base for signaling substances) (binding group) Thiol group, dithio group, pyridine dithio group Thiol group, dithio group, pyridine dithio group disulfide covalent bond dihydroxyboronyl glycosyl cyclic esters of borate covalent bond dihydroxyboronic cis-diol group cyclic esters of borate covalent bond carbonyl group, aldehyde group amino imine bond group covalent bond amino carbonyl group, aldehyde group carboxyl hydroxyl Carboxylic acid ester group covalent bond hydroxyl Carboxyl / carboxylic acid active ester group carboxyl Thiol group Carboxylic acid thioester group covalent bond Thiol group Carboxyl / carboxylic acid active ester group Aminooxy Carboxyl and aldehyde groups Oxygen covalent bond carbonyl group, aldehyde group Aminooxy Carboxyl and aldehyde groups hydroxyl acetal group covalent bond hydroxyl Carboxyl and aldehyde groups

[0029] [Table 1A-2] Metal complex (Ni-NTA (derived from nickel hyponitrotriacetate), etc.) Polyhistamine (His) tag (6×His, 8×His, 10×His, etc.) Coordinate key non-covalent bond basic group (Amine, cyclic secondary amine groups (e.g., pyrrolidyl), piperidinyl, pyridinyl, imidazole, guanidine, etc.) acidic group (Carboxyl / carboxylic acid active ester groups, etc.) hydrogen bonds non-covalent bond hydroxyl acidic group basic group non-covalent bond (Carboxyl groups, etc.) (amino group, cyclic secondary amino group) (e.g., pyrrolidyl, piperidinyl), pyridyl, imidazole, guanidine, etc.), hydroxyl Aromatic base (aminophenyl and other phenyl groups, aminonaphthyl and other naphthyl groups, pyridyl groups, etc.) Aromatic base (aminophenyl and other phenyl groups, aminonaphthyl and other naphthyl groups, pyridyl groups, etc.) hydrophobic key non-covalent bond acetic acid carbonyl group, aldehyde group hydrazone group covalent bond carbonyl group, aldehyde group acetic acid Avidin (neutral avidin, streptoavidin) Biotin (also known as biotin, debiotin) Anti-Biotin-Biotin Bond non-covalent bond Biotin (also known as biotin, debiotin) Avidin (neutral avidin, streptoavidin) Glutathione (GSH) Glutathione S-transferase (GST) Glutathione (GSH)-Glutathione-S-transferase non-covalent bond

[0030] [Table 1A-3] Glutathione S-transferase (GST) Glutathione (GSH) (GST) key Phos tags phosphate ions Coordinate key non-covalent bond phosphate ions Phos tags

[0031] In this specification, "polymer matrix" refers to the polymer forming the matrix, generally referring to a substance formed by the polymerization of monomers. Advantageously, the matrix can be of any shape or structure, as long as it has a shape and structure suitable for the sensor when disposed in the analytical sensor or substrate for the analytical sensor of the present invention. The shape or structure of the matrix may be, for example, thin film or spherical (particle-like). As a preferred composition of the matrix, in order to suppress the adsorption of components other than the target as much as possible, the main component of the matrix is ​​highly biocompatible.

[0032] In this specification, "molecular imprinting polymer" means any polymer used in molecular imprinting technology (see Takeuchi. T et. al. Chromatography, 2016, 37 (2), 43-64.), preferably a material recognition material having a binding space for a target substance obtained by polymerizing a composite of the target substance or its derivative and a functional monomer using covalent and / or non-covalent bonds, along with a crosslinking agent, and then removing the target substance.

[0033] In this specification, "having...at least a portion of an adaptable object" refers to a shape and / or structure that, in the case of an analytical sensor, enables substantial detection or analysis of object molecules. In the case of an analytical sensor of the present invention, it refers to a shape in which object molecules can interact within the recess. If a polymer matrix is ​​formed, theoretically, the optimal shape size may exist within a film thickness range corresponding to less than half the height of the particle nucleus, but it is not desirable to be limited by theory.

[0034] In this specification, "recess" refers to a gap or pore formed to capture a target substance when used in the analytical sensor of the present invention, and preferably refers to a pore formed on the polymer matrix formed in the analytical sensor.

[0035] In this specification, "binding group" refers to a group that can bind to a substance. For example, in this specification, binding groups for signaling substances and binding groups for specific binding molecules may be used.

[0036] In this specification, "a binding group for a signal substance" refers to a group capable of modifying a signal substance. Examples of signal substances include fluorescent molecules, substances containing radioactive elements, and magnetic substances. From the viewpoint of ease of detection, fluorescent substances are preferred as signal substances. Examples of binding groups for signal substances include the binding functional groups listed in Table 2A, 2-1.

[0037] [Table 2A-1] 2-1 2-2 Binding functional groups Corresponding binding functional groups (A binding group for molecules that specifically bind to the target molecule) (binding group) (Binding base of signaling material) (binding group) Amino groups (monovalent amino groups, divalent amino groups, etc.) Carboxylic acid active ester group (Using active ester groups such as N-hydroxybutyric acid diimide, nitrophenol, pentafluorophenol, etc.; active ester groups of aminocarbamates such as NHS aminocarbamate); carboxyl groups; aldehyde groups; isocyanate groups; isothiocyanate groups; epoxy groups; maleimide groups, etc. amino carboxyl carboxyl amino Sugar syl (cis-diol group) dihydroxyboronic dihydroxyboronic Sugar syl (cis-diol group)

[0038] [Table 2A-2] Thiol group Electron-deficient unsaturated carbon groups such as maleimide and acrylate groups; iodoacetamide groups; thiols; pyridine dithiols; olefin (vinyl ethene) / alkynyl groups for free radical addition click chemistry applications; thioester groups for natural chemical linkages, etc. Electron-deficient unsaturated carbon groups such as maleimide and acrylate groups; iodoacetamide groups; thiols; pyridine dithiols; olefin (vinyl ethene) / alkynyl groups for free radical addition click chemistry applications; thioester groups for natural chemical linkages, etc. Thiol group carbonyl group, aldehyde group amino amino carbonyl group, aldehyde group Aminooxy carbonyl group, aldehyde group carbonyl group, aldehyde group Aminooxy carbonyl group, aldehyde group hydroxyl hydroxyl carbonyl group, aldehyde group Hydroxyl group, phenolic hydroxyl group Carboxylic acid active ester group Metal complex (Ni-NTA (derived from nickel hyponitrotriacetate), etc.) Polyhistamine (His) tag (6×His, 8×His, 10×His) acehydrazine carbonyl group, aldehyde group carbonyl group, aldehyde group acetic acid Avidin (neutral avidin, streptoavidin) Biotin (also known as biotin, debiotin) Biotin (also known as biotin, debiotin) Avidin (neutral avidin, streptoavidin) Glutathione (GSH) Glutathione-S-transferase Glutathione-S-transferase Glutathione (GSH)

[0039] In this specification, "binding group for specific binding molecules" refers to a group that can bind to specific binding molecules capable of specifically binding to a target substance. Examples of binding groups for specific binding molecules to a target substance include the binding functional groups listed in Table 3A 2-1.

[0040] [Table 3A-1] 2-1 2-2 Binding functional groups Corresponding binding functional groups (A binding group for molecules that specifically bind to the target molecule) (binding group) (Binding base of signaling material) (binding group) Amino groups (monovalent amino groups, divalent amino groups, etc.) Carboxylic acid active ester group (Using active ester groups such as N-hydroxybutyric acid diimide, nitrophenol, pentafluorophenol, etc.; active ester groups of aminocarbamates such as NHS aminocarbamate); carboxyl groups; aldehyde groups; isocyanate groups; isothiocyanate groups; epoxy groups; maleimide groups, etc. amino carboxyl carboxyl amino Sugar syl (cis-diol group) dihydroxyboronic dihydroxyboronic Sugar syl (cis-diol group) Thiol group Electron-deficient unsaturated carbon groups such as maleimide and acrylate groups; iodoacetamide groups; thiols; pyridine dithiols; olefin (vinyl ethene) / alkynyl groups for free radical addition click chemistry applications; thioester groups for natural chemical linkages, etc. Electron-deficient unsaturated carbon groups such as maleimide and acrylate groups; iodoacetamide groups; thiols; pyridine dithiols; olefin (vinyl ethene) / alkynyl groups for free radical addition click chemistry applications; thioester groups for natural chemical linkages, etc. Thiol group

[0041] [Table 3A-2] carbonyl group, aldehyde group amino amino carbonyl group, aldehyde group Aminooxy carbonyl group, aldehyde group carbonyl group, aldehyde group Aminooxy carbonyl group, aldehyde group hydroxyl hydroxyl carbonyl group, aldehyde group Hydroxyl group, phenolic hydroxyl group Carboxylic acid active ester group Metal complex (Ni-NTA (derived from nickel hyponitrotriacetate), etc.) Polyhistamine (His) tags (6×His, 8×His, 10×His) acehydrazine carbonyl group, aldehyde group carbonyl group, aldehyde group acetic acid Avidin (neutral avidin, streptoavidin) Biotin (also known as biotin, debiotin) Biotin (also known as biotin, debiotin) Avidin (neutral avidin, streptoavidin) Glutathione (GSH) Glutathione-S-transferase Glutathione-S-transferase Glutathione (GSH)

[0042] In this specification, "raw materials for polymer matrix" refers to raw materials that can form a polymer matrix through reaction. Generally, in order to form a polymer matrix, it is sufficient to include at least one monomer and at least one suitable polymerization initiator. In addition, for example, additional monomers (which can be partially polymerized), additional polymerization initiators, crosslinking agents, RAFT (Reversible-addition fragmentation chain transfer) agents, catalysts, reducing agents, or solvents may also be included. Examples of monomers include styrene, N-isopropylacrylamide, 2-methacryloxyethyl phosphocholine, etc., but are not limited to these. Examples of polymerization initiators include 2,2'-azobis(isobutyronitrile) (AIBN), ethyl α-bromoisobutyrate, etc., but are not limited to these. Examples of crosslinking agents include melamine compounds, guanidine compounds, glycourea compounds, N,N'-methylenebisacrylamide, and (tris, tetras, pentas, hexas, poly)ethylene glycol dimethacrylate, but are not limited to these. Examples of RAFT agents include benzyl benzodithioate and 2-cyano-2-[(dodecylthiocarbonyl)thio]propane, but are not limited to these. Examples of catalysts include CuBr2, but are not limited to these. Examples of reducing agents include ascorbic acid, but are not limited to these. Examples of solvents can be selected without particular restriction from commonly known solvents, such as pure water, buffer solution, MeOH, EtOH, DMA (dimethyl acetamide), and DMF (dimethylformamide), but are not limited to these.

[0043] In this specification, "polymer matrix (raw material) is polymerized" means that the polymer matrix (raw material) is polymerized in the presence of the polymer matrix raw material.

[0044] In this specification, "conditions for particle dissociation" refers to conditions under which the substrate and particles can be separated by severing the interaction between the particles disposed on the substrate. Examples include heating, cooling, pH adjustment (acid treatment, alkali treatment), washing with a solution containing surfactants, ultrasonic irradiation, light irradiation, vibration, and reduction treatment.

[0045] In this specification, "substance required for analysis" refers to a substance that can capture and detect the target substance. Examples include capture agents and labels. Capture agents refer to any reagent used to capture the analyte, such as antibodies, antibody fragments, antibody mimics, nucleic acid aptamers (including DNA, RNA, peptide nucleic acids, and artificial nucleic acids), phospholipid recognition proteins, and lectins.

[0046] In this specification, "label" refers to something (e.g., matter, energy, electromagnetic waves, etc.) used to identify target molecules or substances from other sources. Examples of such labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method, etc. In this invention, when labeling is performed using multiple (two or more) labels or factors or methods for capturing them, or fluorescence method, labeling is performed using fluorescent substances with different maximum fluorescence wavelengths. The difference in maximum fluorescence wavelength is preferably 10 nm or more. When labeling ligands, any fluorescent substance can be used as long as it does not affect the function. Examples of fluorescent substances include Alexa TMFluor BODIPY, ATTO, quantum dots (QDot), and fluorescent proteins (GFP (Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), mCherry, etc.). Alexa TMFluor is a water-soluble fluorescent pigment obtained by modifying coumarin, rhodamine, luciferin, anthocyanins, etc. It is a series corresponding to a wide range of fluorescence wavelengths, and compared with other fluorescent pigments of the same wavelength, it is very stable and bright, and also has low pH sensitivity. Examples of combinations of fluorescent pigments with a fluorescence maximum wavelength of 10 nm or higher include: Alexa™ 555 and Alexa™ 633, Alexa™ 488 and Alexa™ 555, etc. When labeling nucleic acids, any pigment can be used as long as it can bind to their base moieties; preferred options include anthocyanins (such as CyDye™ series Cy3, Cy5, etc.), rhodamine 6G reagent, 2-acetaminophen (AAF), AAIF (an iodine derivative of AAF), etc. Examples of fluorescent substances with a maximum fluorescence wavelength difference of 10 nm or more include combinations of Cy5 and rhodamine 6G reagent, combinations of Cy3 and luciferin, and combinations of rhodamine 6G reagent and luciferin. In this invention, such marking can be used to modify the target object so that it can be detected by the detection method used. This modification is well known in the art, and operators can appropriately implement this method based on the marking and the target object.

[0047] In this specification, "coefficient of variation of XX or less" means that the coefficient of variation in the target material detection of the analytical sensor of the present invention is a constant value. The coefficient of variation can be determined based on the standard deviation / mean value. In the present invention, the coefficient of variation can be less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, with less than 20% being a preferred embodiment. The coefficient of variation can be a value within the substrate and / or between substrates, or both.

[0048] In this specification, "at a ratio of YY or higher" means that the analytical sensor of the present invention can be manufactured with a performance value of a certain value or higher at a ratio of YY or higher. In this specification, the performance can be measured, for example, using an automated dispensing device with an attached fluorescence detector (manufactured by SYSTEM INSTRUMENTS Inc.), or using the coefficient of variation in target substance detection. In the present invention, it is preferred that it can be manufactured at a ratio of 80% or higher.

[0049] In this specification, "density of XX to YY particles / mm² on the substrate" refers to the density of particles or recesses disposed on the substrate being in the range of XX to YY particles / mm². In this specification, the density is measured using a fluorescence microscope or a scanning electron microscope.

[0050] In this specification, "spin coating" refers to the following method: using an apparatus that forms a thin film by centrifugal force by rotating a smooth substrate at high speed, a particle dispersion obtained by dispersing particles in a solvent is added to a substrate, and then the substrate is rotated together, thereby coating the particles onto the substrate.

[0051] In this specification, "method of dropping particles onto a substrate" means a method of coating particles onto a substrate by dropping a particle dispersion obtained by dispersing particles in a solvent onto a substrate.

[0052] In this specification, "method of immersing a substrate in a particle dispersion" refers to a method of coating particles onto a substrate by immersing a particle dispersion obtained by dispersing particles in a solvent onto the substrate.

[0053] In this specification, "method of lifting substrate from particle dispersion" refers to a method of coating particles onto a substrate by vertically lifting the substrate from a particle dispersion obtained by dispersing particles in a solvent.

[0054] In this specification, "method of spraying particle dispersion" refers to a method of coating particles onto a substrate by spraying a particle dispersion obtained by dispersing particles in a solvent into a mist onto the substrate.

[0055] In this specification, "signal substance" refers to a substance capable of detecting a target substance and can be used in the same sense as "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.

[0056] In this specification, the term "specificity," when used in relation to affinity, refers to the interaction of a target molecule with a higher affinity than that of other substances. More preferably, it refers to the absence of interaction with substances other than the target molecule. The latter is also referred to as specificity in a narrow sense or selective specificity. Specificity can be determined using various intermolecular interaction analysis devices. For example, it can be measured using various analytical devices using surface plasma resonance (SPR), isothermal titration calorimetry (ITC), or quartz crystal microbalance (QCM).

[0057] (Preferred Embodiment) Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided to provide a more complete understanding of the present invention, and it should be understood that the scope of the present invention should not be limited to the following description. Therefore, it is understood that appropriate changes can be made within the scope of the present invention with reference to the description in this specification. Furthermore, it should be understood that the following embodiments of the present invention can be used alone or in combination.

[0058] <Substrate for Fabricating Analytical Sensors> In one embodiment, the present invention provides a substrate for fabricating analytical sensors, comprising A) a substrate and B) particles monodispersed in the substrate. By manufacturing the substrate in the form of monodispersed particles, the substrate exhibits excellent properties in terms of stability, efficiency, performance, and reproducibility.

[0059] In one embodiment, the present invention provides a substrate for fabricating an analytical sensor, comprising A) a substrate and B) particles disposed on the substrate without aggregation. By manufacturing the substrate in the form of particles disposed without aggregation, the substrate exhibits excellent properties in terms of stability, efficiency, performance, and reproducibility.

[0060] In one embodiment, the present invention provides a substrate for fabricating an analytical sensor, comprising A) a substrate and B) particles disposed in the substrate in a single layer state. By manufacturing the substrate in the form of particles disposed in a single layer state, the substrate exhibits excellent properties in terms of stability, efficiency, performance, and reproducibility.

[0061] In another embodiment, the present invention provides a substrate for fabricating an analytical sensor, comprising A) a substrate and B) particles disposed on the substrate at a density of 1×10³ particles / mm² to 1×10⁶ particles / mm². By manufacturing the substrate with particles disposed at a specific density, the substrate exhibits excellent properties in terms of stability, efficiency, performance, and reproducibility.

[0062] The material used in the substrate of the present invention may be selected from the group consisting of metals, metal oxides, glass, paper (cellulose), cloth, silicon dioxide, silicon and resins, and combinations thereof. Examples of metals include gold, silver, copper, aluminum, titanium, tungsten, and molybdenum. Examples of resins include poly(meth)acrylate, polystyrene, (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.

[0063] The substrate used in this invention may also be formed by combining a plurality of materials selected from the above-mentioned materials. For example, the substrate may also be a metal film disposed on the surface of glass or resin. Furthermore, the shape of the substrate may be plate-shaped or particle-shaped. Preferred examples include gold substrate, glass substrate, gold nanoparticles, silicon dioxide particles (silicon oxide particles, glass beads, etc.).

[0064] The particles used in this invention can be biological molecules or particle nuclei. There are no particular limitations on the particle nucleus as long as it can be used as a template in molecular imprinting; examples include artificially manufactured inorganic and organic particles. Examples of inorganic particles include: metals (gold, silver, platinum, indium tin oxide (ITO), tin oxide (ATO), etc.), metal oxides (iron oxide, aluminum oxide, copper oxide, titanium oxide, zinc oxide, zirconium oxide, cerium oxide, cobalt oxide, etc.), graphene, graphene oxide, carbon nanotubes, diamond nanoparticles, nitrides, fluorides, sulfides, borides, and composite compounds thereof, as well as hydroxyapatite, etc. Silicon dioxide (silicon oxide) is a preferred example. Furthermore, examples of organic particles include: cured latex, dextran, polyglucosamine, polylactic acid, poly(meth)acrylic acid, polymethyl methacrylate, PLGA (poly(lactic-co-glycolic acid) copolymer), polystyrene, and polyethylimide. Additionally, particles with binding functional groups attached to the surface of biological molecules or particle nuclei can also be considered.

[0065] The particle density present on the surface of the substrate of the present invention can be controlled by appropriately adjusting the preparation method when preparing the particles, the solvent used to disperse the particles, the particle concentration, the amount of particle dispersion, and the temperature of the particle dispersion. By controlling the particle density present on the surface of the substrate, the density of the recess of the analytical sensor can also be controlled, and an analytical sensor exhibiting excellent properties in terms of stability, efficiency, and performance can be manufactured.

[0066] In one embodiment, the particles of the present invention are configured with an integral particle of the modification material at a density of approximately 10³ particles / mm² to approximately 10⁸ particles / mm², more preferably approximately 10⁴ particles / mm² to approximately 10⁷ particles / mm², and even more preferably approximately 10⁴ particles / mm² to approximately 10⁶ particles / mm². As upper limits for density, examples include approximately 1×10⁷ particles / mm², approximately 9×10⁶ particles / mm², approximately 8×10⁶ particles / mm², approximately 7×10⁶ particles / mm², approximately 6×10⁶ particles / mm², approximately 5×10⁶ particles / mm², approximately 4×10⁶ particles / mm², approximately 3×10⁶ particles / mm², approximately 2×10⁶ particles / mm², and approximately 1×10⁶ particles / mm². As lower limits, examples include approximately 1×10³ particles / mm², approximately 2×10³ particles / mm², approximately 3×10³ particles / mm², approximately 4×10³ particles / mm², approximately 5×10³ particles / mm², approximately 6×10³ particles / mm², approximately 7×10³ particles / mm², and approximately 8×10⁶ particles / mm². The density configuration is approximately 3 particles / mm², approximately 9 × 10³ particles / mm², approximately 1 × 10⁴ particles / mm², or preferably approximately 10³ particles / mm² to approximately 10¹⁰ particles / mm², more preferably approximately 10⁴ particles / mm² to approximately 10⁹ particles / mm², and even more preferably approximately 10⁴ particles / mm² to approximately 10⁸ particles / mm². As upper limits for density, examples include approximately 1×10¹⁰ particles / mm², approximately 9×10⁹ particles / mm², approximately 8×10⁹ particles / mm², approximately 7×10⁹ particles / mm², approximately 6×10⁹ particles / mm², approximately 5×10⁹ particles / mm², approximately 4×10⁹ particles / mm², approximately 3×10⁹ particles / mm², approximately 2×10⁹ particles / mm², and approximately 1×10⁹ particles / mm². As lower limits, examples include approximately 1×10³ particles / mm², approximately 2×10³ particles / mm², approximately 3×10³ particles / mm², approximately 4×10³ particles / mm², approximately 5×10³ particles / mm², approximately 6×10³ particles / mm², approximately 7×10³ particles / mm², and approximately 8×10⁹ particles / mm². 3 pieces / mm², approximately 9×10³ pieces / mm², approximately 1×10⁴ pieces / mm², etc.

[0067] In one embodiment, the particles used in this invention comprise the substance used in the analytical sensor. The substance used in the analytical sensor may be the substance required for the assay, referring to a substance capable of being detected. Examples include trapping agents, markers, etc. A trapping agent refers to any agent used to trap the analyte, such as antibodies, antibody fragments, antibody-like antibodies, nucleic acid aptamers (including DNA, RNA, peptide nucleic acids, and artificial nucleic acids), phospholipid recognition proteins, and lectins.

[0068] In a preferred embodiment, the particles comprise particles integrated with the modifying substance.

[0069] In another embodiment, in addition to the aforementioned particles and substrate, the present invention further includes C) a polymer matrix, as needed, disposed on the substrate body, and the polymer matrix having at least a portion of recesses for the adaptee, wherein the aforementioned particles are disposed in the recesses. It should be understood that the substrate for fabricating an analytical sensor can be adopted in any of the embodiments described in other parts of this specification, such as <Analytical Sensor> and <Method for Manufacturing Substrate for Fabricating Analytical Sensor>.

[0070] <Analytical Sensor> In another embodiment, the present invention provides an analytical sensor comprising: A) a substrate body; B) a polymer matrix, disposed on the substrate body, wherein the polymer matrix has at least a portion of a recess for an adaptor, and the particle is disposed in the recess; C) a binding base for a signaling substance disposed on the particle; and D) a binding base for a specific binding molecule disposed on the particle to bind with a molecule that is a detection target. It should be understood that the sensor may employ any of the embodiments described in other locations herein, such as substrates for fabricating analytical sensors.

[0071] In one embodiment, the particles used in this invention are preferably disposed on the substrate body at a density of 10³ particles / mm² to 10⁸ particles / mm². More preferably, it is about 10³ particles / mm² to about 10⁷ particles / mm², more preferably about 10⁴ particles / mm² to about 10⁷ particles / mm², and even more preferably about 10⁵ particles / mm² to about 10⁶ particles / mm². As upper limits for density, examples include approximately 1×10⁷ particles / mm², approximately 9×10⁶ particles / mm², approximately 8×10⁶ particles / mm², approximately 7×10⁶ particles / mm², approximately 6×10⁶ particles / mm², approximately 5×10⁶ particles / mm², approximately 4×10⁶ particles / mm², approximately 3×10⁶ particles / mm², approximately 2×10⁶ particles / mm², and approximately 1×10⁶ particles / mm². As lower limits, examples include approximately 1×10³ particles / mm², approximately 2×10³ particles / mm², approximately 3×10³ particles / mm², approximately 4×10³ particles / mm², approximately 5×10³ particles / mm², approximately 6×10³ particles / mm², approximately 7×10³ particles / mm², and approximately 8×10⁶ particles / mm². The density is approximately 3 particles / mm², approximately 9 × 10³ particles / mm², approximately 1 × 10⁴ particles / mm², etc., or preferably 10¹⁰ particles / mm². More preferably, it is arranged in the substrate body at a density of approximately 10³ particles / mm² to approximately 10⁹ particles / mm², more preferably approximately 10⁴ particles / mm² to approximately 10⁸ particles / mm², and even more preferably approximately 10⁵ particles / mm² to approximately 10⁷ particles / mm². As upper limits for density, examples include approximately 1×10¹⁰ particles / mm², approximately 9×10⁹ particles / mm², approximately 8×10⁹ particles / mm², approximately 7×10⁹ particles / mm², approximately 6×10⁹ particles / mm², approximately 5×10⁹ particles / mm², approximately 4×10⁹ particles / mm², approximately 3×10⁹ particles / mm², approximately 2×10⁹ particles / mm², and approximately 1×10⁹ particles / mm². As lower limits, examples include approximately 1×10³ particles / mm², approximately 2×10³ particles / mm², approximately 3×10³ particles / mm², approximately 4×10³ particles / mm², approximately 5×10³ particles / mm², approximately 6×10³ particles / mm², approximately 7×10³ particles / mm², and approximately 8×10⁹ particles / mm². 3 particles / mm², approximately 9 × 10³ particles / mm², approximately 1 × 10⁴ particles / mm², etc. When the density is below this range, it is impossible to perform measurements with sufficient sensitivity to detect the object. When the density is above this range, particles may aggregate, making it impossible to manufacture a sensor with good reproducibility. Examples of preferred densities for monodisperse or non-aggregated recesses include 10⁴ particles / mm² to 10⁶ particles / mm².

[0072] <Substrate for Fabricating Analytical Sensors and Method for Manufacturing Analytical Sensors> In another embodiment, the present invention provides a method for manufacturing a substrate for fabricating analytical sensors. The method includes: A) providing the particles of the present invention; B) adding the particles to the substrate in a monodisperse manner on the substrate body; C) providing, as needed, a raw material for a polymer matrix to the substrate on which the particles are fixed; D) forming, as needed, a substrate having the polymer matrix disposed thereon by providing conditions for polymerization of the substrate with the polymer matrix; and E) forming recesses by providing conditions for the particles to dissociate from the substrate.

[0073] In another embodiment, the present invention provides a method for manufacturing a substrate for fabricating an analytical sensor. The method includes: A) providing the particles of the present invention; B) adding the particles to the substrate in a manner that prevents the particles from agglomerating and disposing them on the substrate body; C) providing, as needed, a raw material for a polymer matrix to the substrate on which the particles are fixed; D) forming, as needed, a substrate having the polymer matrix disposed thereon by providing the substrate with conditions for polymerization of the polymer matrix; and E) forming recesses by providing conditions for the particles to dissociate from the substrate.

[0074] In another embodiment, the present invention provides a method for manufacturing a substrate for fabricating an analytical sensor. The method includes: A) providing the particles of the present invention; B) adding the particles to the substrate in a monodisperse manner on the substrate body; C) providing, as needed, a raw material for a polymer matrix to the substrate on which the particles are fixed; D) forming, as needed, a substrate having the polymer matrix disposed thereon by providing the substrate with conditions for polymerization of the polymer matrix; and E) forming recesses by providing conditions for the particles to dissociate from the substrate.

[0075] In this invention, the step of adding the particles in a monodisperse and / or non-agglomerated manner, and / or in a monolayer configuration, is preferably to add the particles in a monodisperse and / or non-agglomerated manner, and / or in a monolayer configuration, so that the particles maintain their state even after the polymer matrix has polymerized. By providing appropriate modifying substances and / or setting appropriate conditions, the dispersibility can be appropriately maintained or changed, thereby maintaining the state even after the polymer matrix has polymerized. Operators can implement this appropriately based on the examples and other descriptions in this specification.

[0076] Specifically, in this invention, the step of adding particles in a monodisperse and / or non-aggregate manner, and / or in a monolayer manner, includes: adding particles to the substrate at a concentration of 10×10⁰ particles / μL to 2.5×10¹⁰ particles / μL, or at an amount of 1.0×10⁰ particles / mm² to 2.5×10¹⁰ particles / mm² relative to the surface area of ​​the substrate. As upper limits for concentration, examples include: approximately 1×10¹⁰ cells / μL, approximately 9×10⁹ cells / μL, approximately 8×10⁹ cells / μL, approximately 7×10⁹ cells / μL, approximately 6×10⁹ cells / μL, approximately 5×10⁹ cells / μL, approximately 4×10⁹ cells / μL, approximately 3×10⁹ cells / μL, approximately 2×10⁹ cells / μL, approximately 1×10⁹ cells / μL, approximately 9×10⁸ cells / μL, approximately 8×10⁸ cells / μL, approximately 7×10⁸ cells / μL, approximately 6×10⁸ cells / μL, approximately 5×10⁸ cells / μL, approximately 4×10⁸ cells / μL, approximately 3×10⁸ cells / μL, approximately 2×10⁸ cells / μL, approximately 1 ... 8 cells / μL, approximately 9×10⁷ cells / μL, approximately 8×10⁷ cells / μL, approximately 7×10⁷ cells / μL, approximately 6×10⁷ cells / μL, approximately 5×10⁷ cells / μL, approximately 4×10⁷ cells / μL, approximately 3×10⁷ cells / μL, approximately 2×10⁷ cells / μL, approximately 1×10⁷ cells / μL, approximately 9×10⁶ cells / μL, approximately 8×10⁶ cells / μL, approximately 7×10⁶ cells / μL, approximately 6×10⁶ cells / μL, approximately 5×10⁶ cells / μL, approximately 4×10⁶ cells / μL, approximately 3×10⁶ cells / μL, approximately 2×10⁶ cells / μL, approximately 1×10⁶ cells / μL, approximately 9×10⁵ cells / μL, approximately 8×10⁷ ... 5 cells / μL, approximately 7×10⁵ cells / μL, approximately 6×10⁵ cells / μL, approximately 5×10⁵ cells / μL, approximately 4×10⁵ cells / μL, approximately 3×10⁵ cells / μL, approximately 2×10⁵ cells / μL, approximately 1×10⁵ cells / μL, etc., are used as lower limits.Examples include: approximately 1×10⁰ cells / μL, approximately 2×10⁰ cells / μL, approximately 3×10⁰ cells / μL, approximately 4×10⁰ cells / μL, approximately 5×10⁰ cells / μL, approximately 6×10⁰ cells / μL, approximately 7×10⁰ cells / μL, approximately 8×10⁰ cells / μL, approximately 9×10⁰ cells / μL, approximately 1×10¹ cells / μL, approximately 2×10¹ cells / μL, approximately 3×10¹ cells / μL, approximately 4×10¹ cells / μL, approximately 5×10¹ cells / μL, approximately 6×10¹ cells / μL, approximately 7×10¹ cells / μL, approximately 8×10¹ cells / μL, approximately 9×10¹ cells / μL, approximately 1×10² cells / μL, approximately 2×10¹ cells / μL, etc. 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 particles / μL, approximately 5 × 10⁴ particles / μL, approximately 6 × 10⁴ particles / μL, approximately 7 × 10⁴ particles / μL, approximately 8 × 10⁴ particles / μL, approximately 9 × 10⁴ particles / μL, approximately 1 × 10⁵ particles / μL, etc. These represent the upper limit of the amount on the substrate per unit surface area of ​​mm².For example, we can list: approximately 1×10⁻¹⁰ pieces / mm², approximately 9×10⁻⁹ pieces / mm², approximately 8×10⁻⁹ pieces / mm², approximately 7×10⁻⁹ pieces / mm², approximately 6×10⁻⁹ pieces / mm², approximately 5×10⁻⁹ pieces / mm², approximately 4×10⁻⁹ pieces / mm², approximately 3×10⁻⁹ pieces / mm², approximately 2×10⁻⁹ pieces / mm², approximately 1×10⁻⁹ pieces / mm², approximately 9×10⁻⁸ pieces / mm², approximately 8×10⁻⁸ pieces / mm², approximately 7×10⁻⁸ pieces / mm², approximately 6×10⁻⁸ pieces / mm², approximately 5×10⁻⁸ pieces / mm², approximately 4×10⁻⁸ pieces / mm², approximately 3×10⁻⁸ pieces / mm², approximately 2×10⁻⁸ pieces / mm², approximately 1 ... 8 pieces / mm², approx. 1×10⁸; 8 pieces / mm², approx. 9×10⁸; 7 pieces / mm², approx. 8×10⁸; 7 pieces / mm², approx. 7×10⁸; 7 pieces / mm², approx. 6×10⁸; 7 pieces / mm², approx. 5×10⁸; 7 pieces / mm², approx. 4×10⁸; 7 pieces / mm², approx. 3×10⁸; 7 pieces / mm², approx. 2×10⁸; 7 pieces / mm², approx. 1×10⁸; 7 pieces / mm², approx. 9×10⁶; 6 pieces / mm², approx. 8×10⁶; 6 pieces / mm², approx. 7×10⁶; 6 pieces / mm², approx. 6×10⁶; 6 pieces / mm², approx. 5×10⁶; 6 pieces / mm², approx. 4×10⁶; 6 pieces / mm², approx. 3×10⁶; 6 pieces / mm², approx. 2×10⁶; 6 pieces / mm², approx. 2×10⁶ 2. Approximately 1×10⁶ pieces / mm², 2. Approximately 9×10⁵ pieces / mm², 2. Approximately 8×10⁵ pieces / mm², 2. Approximately 7×10⁵ pieces / mm², 2. Approximately 6×10⁵ pieces / mm², 2. Approximately 5×10⁵ pieces / mm², 2. Approximately 4×10⁵ pieces / mm², 2. Approximately 3×10⁵ pieces / mm², 2. Approximately 2×10⁵ pieces / mm², 2. Approximately 1×10⁵ pieces / mm², etc., are used as lower limits.For example, we can list: approximately 1×10⁰ pieces / mm², approximately 2×10⁰ pieces / mm², approximately 3×10⁰ pieces / mm², approximately 4×10⁰ pieces / mm², approximately 5×10⁰ pieces / mm², approximately 6×10⁰ pieces / mm², approximately 7×10⁰ pieces / mm², approximately 8×10⁰ pieces / mm², approximately 9×10⁰ pieces / mm², approximately 1×10¹ pieces / mm², approximately 2×10¹ pieces / mm², approximately 3×10¹ pieces / mm², approximately 4×10¹ pieces / mm², approximately 5×10¹ pieces / mm², approximately 6×10¹ pieces / mm², approximately 7×10¹ pieces / mm², approximately 8×10¹ pieces / mm², approximately 9×10¹ pieces / mm², approximately 1 ... 2 pieces / mm 2, approx. 2×10 2 pieces / mm 2, approx. 3×10 2 pieces / mm 2, approx. 4×10 2 pieces / mm 2, approx. 5×10 2 pieces / mm 2, approx. 6×10 2 pieces / mm 2, approx. 7×10 2 pieces / mm 2, approx. 8×10 2 pieces / mm 2, approx. 9×10 2 pieces / mm 2, approx. 1×10 3 pieces / mm 2, approx. 2×10 3 pieces / mm 2, approx. 3×10 3 pieces / mm 2, approx. 4×10 3 pieces / mm 2, approx. 5×10 3 pieces / mm 2, approx. 6×10 3 pieces / mm 2, approx. 7×10 3 pieces / mm 2, approx. 8×10 3 pieces / mm 2, approx. 9×10 3 pieces / mm 2, approx. 1×10 4 pieces / mm 2. Approximately 2 × 10⁴ pieces / mm², approximately 3 × 10⁴ pieces / mm², approximately 4 × 10⁴ pieces / mm², approximately 5 × 10⁴ pieces / mm², approximately 6 × 10⁴ pieces / mm², approximately 7 × 10⁴ pieces / mm², approximately 8 × 10⁴ pieces / mm², approximately 9 × 10⁴ pieces / mm², approximately 1 × 10⁵ pieces / mm², etc. As one embodiment, the upper limit can be 1.0 × 10¹⁰ pieces / (μL·mm²) to 1.0 × 10⁴ pieces / (μL·mm²), and the lower limit can be 1.0 × 10⁰ pieces / (μL·mm²) to 1.0 × 10⁴ pieces / (μL·mm²).

[0077] In this invention, the step of placing the particles on the substrate can be carried out by any method, such as by dispersing the particles of this invention in a solution and dropping them onto the substrate, and then allowing them to stand or by spin coating.

[0078] In this invention, the step of providing the polymer matrix raw material to the substrate on which the particles are fixed can be carried out by any method, for example by adding a polymeric monomer and providing the particles or polymeric functional groups derived from functional monomers and polymeric monomers as the matrix.

[0079] In this invention, any substance may be used as the raw material for the polymer matrix, provided it is suitable for the desired analysis. Generally, to form the polymer matrix, it is sufficient for the raw material to contain at least one monomer and at least one suitable polymerization initiator. In addition, it may contain, for example, additional monomers (which may be partially polymerized), additional polymerization initiators, crosslinking agents, RAFT agents, catalysts, reducing agents, or solvents. Examples of monomers include styrene, N-isopropylacrylamide, and 2-methacryloxyethyl phosphocholine, but are not limited to these. Examples of polymerization initiators include 2,2'-azobis(isobutyronitrile) (AIBN) and ethyl α-bromoisobutyrate, but are not limited to these. Examples of crosslinking agents include melamine compounds, guanidine compounds, glycourea compounds, N,N'-methylenebisacrylamide, and (tris, tetras, penta, hexa, poly)ethylene glycol dimethacrylate, but are not limited to these. Examples of RAFT agents include benzyl benzodithioate and 2-cyano-2-[(dodecylthiocarbonyl)thio]propane, but are not limited to these. Examples of catalysts include CuBr2, but are not limited to these. Examples of reducing agents include ascorbic acid, but are not limited to these. Examples of solvents commonly known as solvents are not particularly limited, such as pure water, buffer solutions, MeOH, EtOH, DMA, and DMF, but are not limited to these. It should be understood that the particles, and the steps of dispersing and / or dispersing the particles in a monodisperse and / or non-aggregate manner, and / or dispersing them in a monolayer on the substrate, may be carried out in other places in this specification, such as any of the embodiments described in the items <Analytical Sensors> <Method for Manufacturing Substrates for Manufacturing Analytical Sensors>, etc.

[0080] In this invention, the step of forming a substrate having the polymer matrix by subjecting the substrate to polymerization conditions in a polymer matrix can be carried out by any method, such as adding a polymerizable monomer, using particles or polymerizable functional groups derived from functional monomers and polymerizable monomers as a matrix, and using a polymerization initiating group as a polymerization initiator to synthesize a molecularly imprinted polymer located on a local surface of the particles of this invention. This allows a polymer matrix with recesses to be formed on the surface of the substrate.

[0081] In this invention, the step of forming the recess by providing conditions for the particle to dissociate from the substrate can be carried out by any step, for example, by breaking the bonds of the reversible bonding base to remove the particle of this invention. Several examples in the table below have been demonstrated in embodiments of this invention. [Table 11A-1] 1-1 1-2 1-3 1-4 Binding functional groups Corresponding binding functional groups Reversible linker Reversible key style (A binding group for molecules that specifically bind to the target molecule) (binding group) (Reversible linker) (Binding base for signaling substances) (binding group) Thiol group, dithio group, pyridine dithio group Thiol group, dithio group, pyridine dithio group disulfide covalent bond dihydroxyboronic glycosyl cyclic esters of borate covalent bond dihydroxyboronic cis-diol group cyclic esters of borate covalent bond carbonyl group, aldehyde group amino imine bond group covalent bond amino carbonyl group, aldehyde group carboxyl hydroxyl Carboxylic acid ester group covalent bond hydroxyl Carboxyl / carboxylic acid active ester group carboxyl Thiol group Carboxylic acid thioester group covalent bond Thiol group Carboxyl / carboxylic acid active ester group Aminooxy Carboxyl and aldehyde groups Oxygen covalent bond carbonyl group, aldehyde group Aminooxy Carboxyl and aldehyde groups hydroxyl acetal group covalent bond hydroxyl Carboxyl and aldehyde groups

[0082] [Table 11A-2] Metal complex (Ni-NTA (derived from nickel hyponitrotriacetate), etc.) Polyhistamine (His) tag (6×His, 8×His, 10×His, etc.) Coordinate key non-covalent bond basic group (Amine, cyclic secondary amine groups (e.g., pyrrolidyl), piperidinyl, pyridinyl, imidazole, guanidine, etc.) acidic group (Carboxyl / carboxylic acid active ester groups, etc.) hydrogen bonds non-covalent bond hydroxyl acidic group basic group non-covalent bond (Carboxyl groups, etc.) (amino group, cyclic secondary amino group) (e.g., pyrrolidyl, piperidinyl), pyridyl, imidazole, guanidine, etc.), hydroxyl Aromatic base (aminophenyl and other phenyl groups, aminonaphthyl and other naphthyl groups, pyridyl groups, etc.) Aromatic base (aminophenyl and other phenyl groups, aminonaphthyl and other naphthyl groups, pyridyl groups, etc.) hydrophobic key non-covalent bond acetic acid carbonyl group, aldehyde group hydrazone group covalent bond carbonyl group, aldehyde group acetic acid Avidin (neutral avidin, streptoavidin) Biotin (also known as biotin, debiotin) Anti-Biotin-Biotin Bond non-covalent bond Biotin (also known as biotin, debiotin) Avidin (neutral avidin, streptoavidin) Glutathione (GSH) Glutathione S-transferase (GST) Glutathione (GSH)-Glutathione-S-transferase non-covalent bond

[0083] [Table 11A-3] Glutathione S-transferase (GST) Glutathione (GSH) (GST) key Phos tags phosphate ions Coordinate key non-covalent bond phosphate ions Phos tags

[0084] In another embodiment, the present invention provides a method for manufacturing an analytical sensor. The method includes: A) a step of providing particles; B) a step of adding the particles to a substrate in a monodisperse manner on the substrate body; C) a step of providing, if necessary, a raw material for a polymer matrix to the substrate on which the particles are fixed; D) a step of forming a substrate having the polymer matrix disposed thereon by subjecting the substrate to polymerization conditions on the polymer matrix, if necessary; E) a step of forming recesses by subjecting the particles to dissociation from the substrate; and F) a step of incorporating an analytically desired substance into the recesses. It should be understood that the particles, the step of monodispersing the particles on the substrate, the polymerization conditions of the polymer matrix, and the particle dissociation conditions can be implemented in any of the other locations described in this specification, such as <Analytical Sensor> and <Substrate for Manufacturing Analytical Sensors>.

[0085] In another embodiment, the present invention provides a method for manufacturing an analytical sensor. The method includes: A) a step of providing particles; B) a step of adding the particles to a substrate in a manner that prevents particle aggregation and allows them to be disposed on the substrate body; C) a step of providing, if necessary, a raw material for a polymer matrix to the substrate on which the particles are fixed; D) a step of forming a substrate with the polymer matrix disposed thereon by providing the substrate with conditions for polymerization of the polymer matrix, if necessary; E) a step of forming recesses by providing conditions for the particles to dissociate from the substrate; and F) a step of binding the analytical material to the recesses. It should be understood that the particles, the step of preventing particle aggregation and dissociating them on the substrate, the conditions for polymerization of the polymer matrix, and the conditions for particle dissociation can be implemented in any of the embodiments described in other places in this description, such as <Analytical Sensor> and <Substrate for Manufacturing Analytical Sensor>.

[0086] In another embodiment, the present invention provides a method for manufacturing an analytical sensor. The method includes: A) a step of providing particles; B) a step of adding the particles to a substrate in a monolayer configuration on a substrate body; C) a step of providing, if necessary, a raw material for a polymer matrix to the substrate on which the particles are fixed; D) a step of forming a substrate having the polymer matrix configured thereon by providing conditions for polymerization of the substrate with the polymer matrix, if necessary; E) a step of forming recesses by providing conditions for the particles to dissociate from the substrate; and F) a step of incorporating an analytically desired substance into the recesses. It should be understood that the particles, the step of configuring the particles in a monolayer configuration on the substrate, the conditions for polymerization of the polymer matrix, and the conditions for particle dissociation can be implemented in any of the embodiments described in other places in this description, such as <Analytical Sensor> and <Substrate for Manufacturing Analytical Sensors>.

[0087] In one embodiment, the step of binding the substance to be analyzed to the above-mentioned recess can be carried out by any method, for example, by modifying the reversible linker of the broken bond generated in the recess by reduction with a binding base for a specific binding molecule and a binding base for a signaling substance.

[0088] In one embodiment, the present invention provides a method for manufacturing the analytical sensor of the present invention, which involves forming recesses with a density of 2.0 × 10⁷ recesses / mm² or less to produce the aforementioned analytical sensor with a coefficient of variation of a certain reference value (e.g., 20%) or less. The coefficient of variation is a value existing within the substrate and between the substrates, and it is sufficient that the value at at least any location is 20% or less, preferably both are 20% or less.

[0089] The particle density in the step of configuring the particles monodisperse and non-aggregating, and / or configuring them in a monolayer, can be about 10³ particles / mm² to about 10⁸ particles / mm², preferably about 10³ particles / mm² to about 10⁷ particles / mm², more preferably about 10⁴ particles / mm² to about 10⁷ particles / mm², or it can be about 10³ particles / mm² to about 10¹⁰ particles / mm², preferably about 10³ particles / mm² to about 10⁹ particles / mm², more preferably about 10⁴ particles / mm² to about 10⁸ particles / mm². In one embodiment, the step of configuring the particles monodisperse and / or non-aggregating, and / or configuring them in a monolayer includes the step of configuring the particles of the present invention on a substrate at a density of 10³ particles / mm² to 10¹⁰ particles / mm². An ideal density of particles disposed on a substrate can be exemplified as 10⁴ particles / mm² to 10⁷ particles / mm². This density can be confirmed based on surface observation using a fluorescence microscope or a scanning electron microscope.

[0090] In one embodiment, a method is provided to provide, during the step of forming the recesses, the monodisperse or non-agglomerated recesses of the present invention having a density of 10³ recesses / mm² to 10¹⁰ recesses / mm². A preferred density of recesses disposed on a substrate is exemplified as 10³ recesses / mm² to 10⁷ recesses / mm². Furthermore, this density can be confirmed based on surface observation using a fluorescence microscope or a scanning electron microscope.

[0091] In one embodiment, the steps of configuring the particles as monodisperse and / or non-aggregate, and / or as a single layer, can be carried out by any method, including, for example, configuring the particles by spin coating, drop onto a substrate, immersion in a particle dispersion, lifting the substrate from a particle dispersion, spraying (coating) the particles, or printing with an inkjet printer.

[0092] (Example of Manufacturing an Analytical Sensor) The steps for manufacturing an analytical sensor can be performed as follows: The particles of the present invention are dispersed in a solution, dropped onto a substrate, and then allowed to stand or spin-coated. A polymerizable monomer is added, and the particles or polymerizable functional groups derived from functional monomers and polymerizable monomers are provided as a matrix. A molecularly imprinted polymer located on a local surface of the particles of the present invention is synthesized using a polymerization initiator. This forms a polymer matrix with recesses on the surface of the substrate. The particles of the present invention are removed by weakening (or dissociating) the interaction between the particle nucleus and the modifying substance. The reversible linkers present (or exposed) in the recesses are modified with binding groups for specific binding molecules and binding groups for signaling substances.

[0093] (Application) The analytical sensor of the present invention can be used to detect the sensing of an object. As a more specific application, it can be determined according to the type of specific binding base, for example, it can be used for diagnostic purposes or treatment monitoring based on renal function, liver function, presence or degree of inflammation, presence or degree of tumor, etc.

[0094] The analytical sensor of the present invention can be used for the following purposes.

[0095] It can be used for: in vivo / in vitro imaging or therapeutic purposes of the test subject, virus sensing in the human body or environment, and analysis of food, crops, livestock, etc.

[0096] The present invention has been described above with reference to preferred embodiments for ease of understanding. Hereinafter, the present invention will be described based on embodiments; however, the above description and the following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention. Therefore, the scope of the present invention is not limited to the embodiments specifically described in this specification, nor to the embodiments, but only to the scope of the claims. [Examples]

[0097] In this embodiment, examples related to the manufacture and use of the compounds of the present invention are described. Furthermore, cell microscopy was performed using a CKX31 (OLYMPUS, Tokyo, Japan), centrifuges were used using a KUBOTA 2800 (KUBOTA, Tokyo, Japan), incubators were used using a CO2 WATER JACKETED INCUBATOR (Thermo Fisher Scientific Inc, Massachusetts, USA), autoclaves were used using a KS-243 (TOMY SEIKO Co, Ltd., Tokyo, Japan), and cleaning stations were used using a sterile cleaning station (ORIENTAL GIKEN INC, Tokyo, Japan). Extracellular body concentration was measured using qNano Gold (Izon Science Ltd., Christchurch, New Zealand). Furthermore, UV ozone treatment of the gold substrate was performed using a UV Ozone Cleaner (BioForce Nanosciences, Inc.), and fluorescence measurements were performed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with an automated SIC dispensing device (SYSTEM INSTRUMENTS Co., Ltd., Tokyo, Japan) and Andor SOLIS (Andor Technology Ltd, Belfast, Northern Ireland) as the optical separation software.

[0098] Furthermore, MALDI-TOF-MS (Matrix Assisted Laser Desorption / Ionization-Time of Flight-Mass Spectrometry) was performed using a MALDI-TOF / MS (MALDI-7090, Shimadzu Corporation), the analysis software was (MALDI Solutions, Shimadzu Corporation), and calibration was performed using protein calibration standard I (Bruker Corporation). Sinapic acid was used as the matrix. CD spectroscopy was performed using a J-725 circular dichroism dispersive meter (Japan Spectrophotometer, Tokyo, Japan). To prepare the buffer solution, the pH was measured using a benchtop pH meter F-52 (HORIBA, Kyoto, JAPAN). Fluorescence spectroscopy was performed using a Hitachi Advanced Technology Fluorescence Spectrophotometer F-2500 (Tokyo, Japan). The ultrafiltration membrane used for ultrafiltration was an Amicon Ultra-4 (10kDa). Absorption spectroscopy measurements were performed using a Thermo Scientific™ Nanodrop™ One ultra-micro UV-Vis spectrophotometer (Thermo Fisher). The average particle size and polydispersity index (PDI) of the prepared particles were measured using a Zetasizer NANO-ZS MAL500735 (Malvern, UK) via dynamic light scattering (DLS), with Zetasizer software used for analysis. TEM analysis was performed using a transmission electron microscope (JEM-1230, NEC).

[0099] (Example 1: Synthesis of Modified Substances) 1. Experimental Procedure 1-1. Polymer Synthesis

[0100] [Chemical 1] Synthesis of various cationic polymers

[0101] 1-1-1. Synthesis of Polymer E2

[0102] [Chemical Engineering 2] Process 1. Polymer E2 Synthesis Process

[0103] The reagents of the formulation shown in Table 1-1 were dissolved in DMF (2 mL), and after degassing with argon, the polymerization reaction was carried out in an oil bath (75°C) for 24 hours. The reaction solution was cooled to allow it to come into contact with air, thereby stopping the reaction. The reaction solution was then dropped into diethyl ether to generate a precipitate, which was then recovered. This operation was repeated twice. The recovered precipitate was vacuum dried to obtain polymer (E2-0). (Yield: 100 mg) 100 mg of the obtained polymer was dispersed in a dichloromethane / dimethyl ether = 1 / 1 (v / v) solution (10 mL), and 92 mg (0.5 mmol) of N-butadiene methacrylate and 140 μL (1.0 mmol) of triethylamine (TEA) were added. The mixture was stirred at room temperature for 24 hours. The solvent after the reaction was removed by vacuum distillation using an evaporator. A small amount of dichloromethane was added to the residue, followed by the addition of excess hexane to produce a precipitate. The precipitate was recovered (this operation was repeated twice). The obtained precipitate was dried under vacuum to obtain polymer E2. (Yield: 101 mg) The estimated composition of the obtained polymer E2 was determined based on 1H-NMR (DMSO-d6) (AVANCE-500, Bruker). (Figure 1-1)

[0104] [Table 1-1] Table 1-1 Synthesis Formulation of Polymer E2 Sample concentration main raw materials N-Isopropylacrylamide (NIPAm) 300 mM cationic portion N-(3-Dimethylaminopropyl)methacrylamide hydrochloride 100 mM Modified areas Cys-methacrylamide hydrochloride 100 mM RAFT agent 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 12.5 mM Initiator 2,2'-Azobis(isobutyronitrile)(AIBN) 6.25 mM solvent DMF 2 mL

[0105] 1-1-2. Synthesis of Polymer E3

[0106] [Chemical 3] Process 2. Polymer E3 Synthesis Process

[0107] The reagents of the formulation shown in Table 1-2 were dissolved in DMF (2 mL), degassed with argon, and then subjected to polymerization in an oil bath (75°C) for 24 hours. The reaction solution was cooled to allow it to come into contact with air, thereby stopping the reaction. The reaction solution was then dropped into diethyl ether to produce a precipitate, which was then recovered. This operation was repeated twice. The recovered precipitate was vacuum dried to obtain the polymer. (Yield: 105 mg) The polymer was dispersed in dichloromethane (10 mL), and 110 mg (0.6 mmol) of N-butadiene methacrylate and 140 μL (1.0 mmol) of triethylamine (TEA) were added. The mixture was stirred at room temperature for 24 hours. Excess hexane was added to the reaction solution to produce a precipitate, which was then recovered (this operation was repeated twice). The obtained precipitate was vacuum dried to obtain polymer E3. (Yield: 115 mg) The obtained polymer was dispersed in dichloromethane (6 mL), and 1.0 mL of dichloromethane containing 4N HCl was added. The mixture was stirred for 2 hours under ice bath cooling. Then, it was stirred overnight at room temperature. Hexane was added to the reaction solution to produce a precipitate, which was recovered. A small amount of dichloromethane was added to the recovered precipitate, followed by the addition of excess hexane to wash the precipitate. The recovered precipitate was dried under vacuum. (Yield: 105 mg) The estimated composition of the obtained polymer E3 was determined based on 1H-NMR (DMSO-d6) (AVANCE-500, Bruker). (Figures 1-2)

[0108] [Table 1-2] Table 1-2 Synthesis Formulation of Polymer E3 Sample concentration main raw materials N-Isopropylacrylamide (NIPAm) 300 mM cationic sites N-Boc-(3-aminopropyl)methacrylamide hydrochloride 100 mM Modified areas Cys-methacrylamide 100 mM RAFT agent 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 12.5 mM Initiator 2,2'-Azobis(isobutyronitrile)(AIBN) 6.25 mM solvent DMF 2 mL

[0109] 1-1-3. Synthesis of Polymer E4

[0110] [Chemical Engineering 4] Process 3. Polymer E4 Synthesis Process

[0111] The reagents of the formulations shown in Table 1-3 were dissolved in DMF (2 mL), and after degassing with argon, a polymerization reaction was carried out in an oil bath (75°C) for 24 hours. The reaction solution was cooled to allow it to come into contact with air, thereby stopping the reaction. The reaction solution was then added dropwise to diethyl ether to form a precipitate, which was then recovered. This operation was repeated twice. The recovered precipitate was vacuum dried to obtain the polymer. (Yield: 70 mg)

[0112] The obtained polymer was dispersed in dichloromethane (10 mL), and 1.0 mL of dichloromethane containing 4N HCl was added. The mixture was stirred for 2 hours under ice bath cooling. Then, it was stirred overnight at room temperature. Hexane was added to the post-reaction solution to produce a precipitate, which was recovered. A small amount of dichloromethane was added to the recovered precipitate, followed by the addition of excess hexane to wash the precipitate. The recovered precipitate was then vacuum dried. Subsequently, the polymer was dispersed in dichloromethane (10 mL), and 73 mg (0.4 mmol) of N-butadiene methacrylate and 84 μL (0.6 mmol) of triethylamine (TEA) were added. The mixture was stirred for 24 hours at room temperature. Excess hexane was added to the post-reaction solution to produce a precipitate, which was recovered (this operation was repeated twice). The obtained precipitate was vacuum dried to obtain E4. (Yield: 62 mg) The estimated composition of the obtained E4 was determined based on 1H-NMR (DMSO-d6) (AVANCE-500, Bruker). (Figures 1-3)

[0113] [Table 1-3] Table 1-3 Synthesis Formulation of Polymer E4 Sample concentration main raw materials N-Isopropylacrylamide (NIPAm) 200 mM cationic sites N-(3-Dimethylaminopropyl)methacrylamide hydrochloride 100 mM Modified areas Cys-methacrylamide hydrochloride 200 mM RAFT agent 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 12.5 mM Initiator 2,2'-Azobis(isobutyronitrile)(AIBN) 6.25 mM solvent DMF 2 mL

[0114] 1-2-1 Synthesis of Polymer F1: The reagents in the formulations shown in Table 1-4 were dissolved in DMF (2 mL), degassed with argon, and then polymerized in an oil bath (75°C) for 24 hours. The reaction solution was cooled to allow it to come into contact with air, thereby stopping the reaction. The reaction solution was then dropped into diethyl ether to produce a precipitate, which was recovered. This operation was repeated twice. The recovered precipitate was vacuum dried to obtain the polymer. (Yield: 105.7 mg) The obtained polymer was dispersed in dichloromethane (5 mL), and 34.1 mg (0.19 mmol) of N-butadiene methacrylate and 38.9 μL (0.28 mmol) of triethylamine (TEA) were added. The mixture was stirred at room temperature for 24 hours. Excess hexane was added to the reaction solution to produce a precipitate, which was recovered (this operation was repeated twice). The obtained precipitate was vacuum dried to obtain F1. (Yield: 19.1 mg) The composition of the obtained F1 was estimated based on 1H-NMR (DMSO-d6) (AVANCE-500, Bruker).

[0115] [Table 1-4] Table 1-4 Synthesis Formulations for Fluorescent Polymer F1 Sample concentration main raw materials N-Isopropylacrylamide (NIPAm) 275 mM cationic sites N-(3-Dimethylaminopropyl)methacrylamide hydrochloride 100 mM Modified areas Cys-methacrylamide hydrochloride 100 mM Fluorescent parts Methacryloxyethylthioamine methyl rhodamine B 25 mM RAFT agent 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 12.5 mM Initiator 2,2'-Azobis(isobutyronitrile): V-60 6.25 mM solvent DMF 2 mL

[0116] Synthesis of Polymer (AN1): 64 mg of Boc-cystamine methacrylamide, 29 μL of tributyl acrylate, and 68 mg of NIPAM were dissolved in 1.8 mL of DMF. Finally, 0.2 mL of DMF containing 8.6 mg of RAFT agent (2-cyano-2-[(dodecylthiocarbonyl)thio]propane) and 2.1 mg of initiator (AIBN) was added for degassing with argon purging to remove dissolved oxygen. The polymerization reaction was then carried out in an oil bath at 75°C for 24 h. After the reaction, the reaction mixture was added dropwise to excess diethyl ether to recover the precipitate. (Yield: 51 mg) The obtained polymer was dissolved in dichloromethane (5 mL) and stirred under ice bath cooling. 1.5 mL of trifluoroacetic acid was added, and the mixture was stirred overnight under light protection. After the reaction, diethyl ether was added to recover the precipitate, which was then washed with diethyl ether. (Yield: 22 mg) The obtained polymer was dispersed in dichloromethane, and 15 mg (0.08 mmol) of N-butadiene methacrylate and 17 μL (0.12 mmol) of triethylamine were added. The mixture was stirred overnight at room temperature. Diethyl ether was added to the reaction solution to recover the precipitate. The precipitate was dissolved in a small amount of dichloromethane, and excess hexane was added to precipitate the precipitate. This operation was repeated twice, followed by vacuum drying to obtain the target polymer (AN1). (Yield: 16 mg)

[0117] 1-1. Synthesis of Polymer (E101) [Chemistry 5] Polymer Formulation: [Table A-1] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 4-[(2-methyl-1-sideoxy-2-propen-1-yl)amino]butyric acid 171.19 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0118] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0119] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 54 mg (0.3 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E101).

[0120] 1-2. Formation and Immobilization of Polymer (E101)-Particle Core Complex on Substrate: An aqueous solution of polymer (E101) (2 mg / mL) was mixed with a particle core dispersion having basic groups on its surface at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and amino-EG6undecylthiol hydrochloride and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain the particle-immobilized substrate.

[0121] 2-1. Synthesis of Polymer (E102) [Chemical Engineering 6] Polymer Formulation: [Table A-2] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 N-Phenylacetylamine 147.17 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0122] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0123] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 54 mg (0.3 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E102).

[0124] 2-2. Formation and Immobilization of Polymer (E102)-Particle Core Complex on Substrate An aqueous solution of polymer (E102) (2 mg / mL) was mixed with a particle core dispersion having hydrophobic groups such as benzene rings on its surface at a ratio of 1 / 1 (v / v). The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified only with 2-(2-bromoisobutyroxy)undecylthiol or a mixture thereof with undecylthiol (SAM, Self-Assembly Monlayer), and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain the particle-immobilized substrate.

[0125] 3-1. Synthesis of Polymer (E103) [Chemistry 7] Polymer Formulation: [Table A-3] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 N-Hexylmethacrylamide 169.26 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0126] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0127] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 54 mg (0.3 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E103).

[0128] 3-2. Formation and Immobilization of Polymer (E103)-Particle Core Complex on Substrate: An aqueous solution of polymer (E103) (2 mg / mL) was mixed with a particle core dispersion having hydrophobic groups such as benzene rings or alkyl chains on its surface at a ratio of 1 / 1 (v / v). The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified only with 2-(2-bromoisobutyroxy)undecylthiol or a mixture thereof with undecylthiol (SAM), and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0129] 4-1. Synthesis of Polymer (E104) [Chemical Engineering 8] Polymer Formulation: [Table A-4] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 NTA monomer (N2,N2-bis(carboxymethyl)-N6-(2-methyl-1-sideoxy-2-propen-1-yl)-L-lysine) 330.33 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0130] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0131] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 54 mg (0.3 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E104).

[0132] 4-2. Formation and Immobilization of Polymer (E104)-Particle Core Complex on Substrate An aqueous solution of polymer (E104) (2 mg / mL) and a particle core dispersion with His-tagged or other coordinate-bonding groups on the surface were mixed at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxyl-EG6undecylthiol was immersed in CH2Cl2 containing 0.1 M EDC and 0.05 M NHS and allowed to stand for 1 hour. The substrate was washed with CH2Cl2 and dried with nitrogen. 4 μL of the polymer (E104)-particle core mixture was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0133] 5-1. Synthesis of Polymer (E105) [Chemical 9] 100 mg of polymer (E101) and 140 μL (1.0 mmol) of TEA were dissolved in CH2Cl2 and stirred under ice bath cooling. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added, and the mixture was stirred overnight. After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH2Cl2, and excess hexane was added to produce another precipitate. The precipitate was recovered and dried under vacuum to obtain polymer (E101)-NHS (E112). 10 mg of the polymer prepared in the previous step and 28 μL of TEA were dissolved in DMF (1 mL) and stirred. 3 mg of a synthetic peptide (with a structure in which the C-terminal carboxyl group is acetylated, having 6 histidine nucleotides bound from the N-terminus, 3 glycine nucleotides bound from the N-terminus, and a lysine nucleotide bound from the C-terminus) was added, and the mixture was stirred overnight. After the reaction, a small amount of CH₂Cl₂ was added, followed by the addition of excess n-hexane to produce a precipitate. The precipitate was recovered, and a small amount of CH₂Cl₂ was added, followed by the addition of excess n-hexane to wash the precipitate. The precipitate was then dried under vacuum to obtain the polymer (E105).

[0134] 5-2. Formation and Immobilization of Polymer (E105)-Particle Core Complex on Substrate An aqueous solution of polymer (E105) (2 mg / mL) and a particle core dispersion with NTA groups or other coordinating groups on the surface were mixed at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6undecylthiol hydrochloride was immersed in DMSO containing 1 mM of N-[5-(4-isothiocyanobenzyl)amino-1-carboxypentyl]iminodiacetic acid and allowed to stand for 1 hour. The substrate was washed with DMSO and EtOH and dried with nitrogen. 4 μL of the polymer (E105)-particle core mixture was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0135] 6-1. Synthesis of Polymer (E106) [Chemistry 10]

[0136] 100 mg of polymer (E101) and 140 μL (1.0 mmol) of TEA were dissolved in CH2Cl2 and stirred while cooling in an ice bath. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added, and the mixture was stirred overnight. After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH2Cl2, and excess hexane was added to produce another precipitate. The precipitate was recovered and dried under vacuum to obtain polymer (E101)-NHS (E112).

[0137] Dissolve 10 mg of the polymer prepared in the previous step in 50 mM carbonate buffer (pH 8.5) (1 mL) and stir. Add 3 mg of avidin and stir overnight. Transfer the reaction solution into a 100 kDa dialysis membrane and dialyze in phosphate buffer (pH 7.4). After dialysis, concentrate by ultrafiltration to obtain a solution of the target polymer (E106).

[0138] 6-2. Formation and Immobilization of Polymer (E106)-Particle Core Complex on Substrate An aqueous solution of polymer (E106) (2 mg / mL) was mixed with a particle core dispersion having biotin or its derivatives on its surface at a 1 / 1 (v / v) ratio. The solution was appropriately diluted with phosphate buffer. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6undecylthiol hydrochloride was immersed in DMSO containing 0.1 M EDC and 0.05 M biotin and allowed to stand for 1 hour. The substrate was washed with DMSO and EtOH after the reaction and dried with nitrogen. 40 μL of a polymer (E105)-particle core mixture was added to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was washed with phosphate buffer to obtain a particle-immobilized substrate.

[0139] 7-1. Synthesis of Polymer (E107) [Chemistry 11] Polymer Formulation: [Table A-5] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 Biotin monomer ((3aS,4S,6aR)-hexahydro-2-sideoxy-N-[3-[(1-sideoxy-2-propen-1-yl)amino]propyl]-1H-thieno[3,4-d]imidazol-4-pentazolamine) 354.47 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0140] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0141] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 54 mg (0.3 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E107).

[0142] 7-2. Formation and Immobilization of Polymer (E107)-Particle Core Complex on Substrate An aqueous solution of polymer (E107) (2 mg / mL) was mixed with a dispersion of avidin-modified particles at a 1 / 1 (v / v) ratio. The solution was appropriately diluted with PBS (Phosphate Buffered Saline). A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxyl-EG6undecylthiol was immersed in CH2Cl2 containing 0.1 M EDC and 0.05 M NHS and allowed to stand for 1 hour. The substrate was washed with CH2Cl2 and dried with nitrogen. A 1 mg / mL avidin solution was added to the substrate and allowed to stand for 1 hour. The substrate was washed with phosphate buffer after the reaction. The polymer (E107)-particle core mixture was added to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was washed with phosphate buffer to obtain a particle-immobilized substrate.

[0143] 8-1. Synthesis of Polymer (E108) [Chemical 12] 100 mg of polymer (E3) and 140 μL (1.0 mmol) of TEA were dissolved in CH2Cl2 and stirred under ice bath cooling. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of 3-mercaptopropionic acid were added, and the mixture was stirred overnight. After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH2Cl2, and excess hexane was added to produce another precipitate. The precipitate was recovered and dried under vacuum to obtain polymer (E108).

[0144] 8-2. Formation and Immobilization of Polymer (E106)-Particle Core Complex on Substrate An aqueous solution of polymer (E108) (2 mg / mL) was mixed with a particle core dispersion having thiol reactive groups such as maleimine and pyridine dithioyl groups on its surface at a 1 / 1 (v / v) ratio. The solution was appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6undecylthiol hydrochloride was immersed in DMF containing 0.1 M EDC and 0.05 M 3-maleimine propionic acid or 3-(2-pyridyldithio)propionic acid and allowed to stand for 1 hour. The substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E108)-particle core mixture was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was cleaned with EtOH to obtain a particle-immobilized substrate.

[0145] 9-1. Synthesis of Polymer (E109) ​​[Chemical 13] 100 mg of polymer (E3) and 140 μL (1.0 mmol) of TEA were dissolved in CH2Cl2 and stirred under ice bath cooling. 40 mg (0.2 mmol) of EDC and 34 mg (0.2 mmol) of 3-maleiminopropionic acid were added, and the mixture was stirred overnight. After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH2Cl2, and excess hexane was added to produce another precipitate. The precipitate was recovered and dried under vacuum to obtain polymer (E108).

[0146] 9-2. Formation and Immobilization of Polymer (E109)-Particle Core Complex on Substrate An aqueous solution of polymer (E109) ​​(2 mg / mL) was mixed with a particle core dispersion having thiol groups on its surface at a 1 / 1 (v / v) ratio. The solution was appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6undecylthiol hydrochloride was immersed in DMF containing 0.1 M EDC and 0.05 M 3-mercaptopropionic acid and allowed to stand for 1 hour. The substrate was washed with EtOH after the reaction and dried with nitrogen. 4 μL of the polymer (E109)-particle core mixture was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0147] 10-1. Synthesis of Polymer (E110) [Chemistry 14] Polymer Formulation: [Table A-6] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 Aldehyde monomer (4-methoxy-N-[2-[(1-sideoxy-2-propen-1-yl)amino]propyl]benzamide) 258.26 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0148] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0149] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 54 mg (0.3 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E110).

[0150] 10-2. Formation and Immobilization of Polymer (E110)-Particle Core Complex on Substrate: An aqueous solution of polymer (E110) (2 mg / mL) was mixed with a particle core dispersion modified with amino groups on the surface at a ratio of 1 / 1 (v / v). The solution was appropriately diluted with DMF. The polymer (E110)-particle core mixture was added dropwise to a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol, and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0151] 11-1. Synthesis of Polymer (E111) [Chemistry 15] Polymer Formulation: [Table A-7] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 Boc-oxime monomer (1,1-dimethyl ethyl 11-methyl-5,10-di-side-oxy-3-oxa-2,6,9-triazadodec-11-enoic acid) 301.34 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0152] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0153] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 54 mg (0.3 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was then redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum.

[0154] The obtained polymer was dissolved in CH₂Cl₂ and stirred while cooled in an ice bath. 1 mL of dialkyl containing 4 N HCl was added, and the mixture was stirred overnight. Hexane was added to the reaction solution to recover the precipitate. A small amount of CH₂Cl₂ was added to the precipitate, followed by the addition of excess hexane to wash the precipitate. The precipitate was then dried under vacuum to obtain the polymer (E111).

[0155] 11-2. Formation and Immobilization of Polymer (E111)-Particle Core Complex on Substrate An aqueous solution of polymer (E111) (2 mg / mL) was mixed with a particle core dispersion modified with aldehyde groups at a ratio of 1 / 1 (v / v). The solution was appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxyl-EG6undecylthiol was immersed in DMF containing 0.1 M EDC and 0.05 M 4-methylbenzoic acid and allowed to stand for 1 hour. The substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E111)-particle core mixture was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0156] 12-1. Synthesis of Polymer (E112) [Chemical 16] 100 mg of polymer (E101) and 140 μL (1.0 mmol) of TEA were dissolved in CH2Cl2 and stirred under ice bath cooling. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added, and the mixture was stirred overnight. After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH2Cl2, and excess hexane was added to produce another precipitate. The precipitate was recovered and dried under vacuum to obtain polymer (E112).

[0157] 12-2. Formation and Immobilization of Polymer (E112)-Particle Core Complex on Substrate An aqueous solution of polymer (E112) (2 mg / mL) was mixed with a particle core dispersion having amine groups on its surface at a 1 / 1 (v / v) ratio. After standing overnight, the solution was diluted 80 times with DMF. 4 μL of the polymer (E112)-particle core mixture was added dropwise to a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and 10-mercapto-N,N,N-trimethyl-1-decammonium chloride, and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0158] 13-1. Synthesis of Polymer (E113) [Chemistry 17] Polymer Formulation: [Table A-8] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 GEMA 292.28 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0159] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0160] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 27 mg (0.15 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E113).

[0161] 13-2. Formation and Immobilization of Polymer (E111)-Particle Core Complex on Substrate An aqueous solution of polymer (E113) (2 mg / mL) was mixed with a particle core dispersion modified with phenylboronic acid groups at a ratio of 1 / 1 (v / v). The solution was appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6undecylthiol hydrochloride was immersed in DMF containing 0.1 M EDC and 0.05 M 4-carboxyphenylboronic acid (or 3-fluoro-4-carboxyphenylboronic acid) and allowed to stand for 1 hour. The substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E113)-particle core mixture was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0162] 14-1. Synthesis of Polymer (E114) [Chemistry 18] Polymer Formulation: [Table A-9] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 4-(2-Methylpropenylaminoethylaminomethyl)-3-fluorophenylboronic acid 292.06 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0163] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0164] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 27 mg (0.15 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E114).

[0165] 14-2. Formation and Immobilization of Polymer (E114)-Particle Core Complex on Substrate An aqueous solution of polymer (E114) (2 mg / mL) was mixed with a particle core dispersion modified with a cis-diol structure at a 1 / 1 (v / v) ratio. The solution was appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6undecylthiol hydrochloride was immersed in DMF containing TEA and α-D-mannopyranosylphenyl isothiocyanate and allowed to stand for 1 hour. The substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E114)-particle core mixture was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0166] 15-1. Synthesis of Polymer (E115) [Chemistry 19] Polymer Formulation: [Table A-10] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 N-Acryloylhomocysteine ​​Thiolactone 171.22 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0167] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0168] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 27 mg (0.15 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E115).

[0169] 15-2. Formation and Immobilization of Polymer (E115)-Particle Core Complex on Substrate An aqueous solution of polymer (E114) (2 mg / mL) was mixed with a particle core dispersion modified with amino groups at a 1 / 1 (v / v) ratio. The solution was appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6undecylthiol hydrochloride was immersed in DMF containing EDC and 3-(2-pyridyldithio)propionic acid and allowed to stand for 1 hour. The substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E115)-particle core mixture was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0170] 16-1. Synthesis of Polymer (E116) [Chemistry 20] Polymer Formulation: [Table A-11] Sample Mw. concentration (mM) Final liquid volume (mL) Cystamine monomer (N-[2-[(2-aminoethyl)dithio]ethyl]-2-methyl-2-propenylamine hydrochloride) 256.82 100 1 3-Azidepropyl methacrylate 169.18 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0171] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon and polymerized in an oil bath (set temperature: 75 °C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum. The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred while cooled in an ice bath. 27 mg (0.15 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight (14 h). After the reaction, n-hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and an excess of n-hexane was added to produce another precipitate. The precipitate was then recovered and dried under vacuum to obtain the polymer (E116).

[0172] 16-2. Formation and Immobilization of Polymer (E116)-Particle Core Complex on Substrate An aqueous solution of polymer (E116) (2 mg / mL) was mixed with a particle core dispersion modified with alkyne groups at a 1 / 1 (v / v) ratio. 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. The solution was appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and amino-EG6undecylthiol hydrochloride was immersed in DMF containing EDC and 4-(3-butyn-1-yldithio)butyric acid and allowed to stand for 1 hour. The substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E116)-particle core mixture (containing copper sulfate and ascorbic acid) was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was cleaned with EtOH to obtain a particle-immobilized substrate.

[0173] 17-1. Synthesis of Polymer (E116) [Chemical 21] Polymer (E112) and 140 μL (1.0 mmol) of TEA were dissolved in CH2Cl2 and stirred under ice bath cooling. 11 mg (0.2 mmol) of propargylamine was added and stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH2Cl2, and excess hexane was added to produce a precipitate. The precipitate was recovered and dried under vacuum to obtain polymer (E117).

[0174] 17-2. Formation and Immobilization of Polymer (E117)-Particle Core Complex on Substrate An aqueous solution of polymer (E117) (2 mg / mL) was mixed with a dispersion of particle cores modified with azide groups at a 1 / 1 (v / v) ratio. 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. The solution was appropriately diluted with DMF. A gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and amino-EG6undecylthiol hydrochloride was immersed in DMF containing EDC and 3-[(2-azidoethyl)dithio]propionic acid and allowed to stand for 1 hour. The substrate was washed with EtOH and dried with nitrogen. 4 μL of the polymer (E117)-particle core mixture (containing copper sulfate and ascorbic acid) was added dropwise to the substrate and allowed to stand for 1 hour. Subsequently, the substrate was cleaned with EtOH to obtain a particle-immobilized substrate.

[0175] 18-1. Synthesis of Polymer (E201) [Chemistry 22] Polymer Formulation: [Table A-12] Sample Mw. concentration (mM) Final liquid volume (mL) Aldehyde monomer (4-methoxy-N-[2-[(1-sideoxy-2-propen-1-yl)amino]propyl]benzamide) 258.26 100 1 N-(3-(dimethylamino)propyl)methacrylamide 170.25 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0176] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0177] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 25 mg (0.2 mmol) of N-(2-aminoethyl)methacrylamide was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E201).

[0178] 18-2. Formation and Immobilization of Polymer (E201)-Particle Core Complex on Substrate: An aqueous solution of polymer (E201) (2 mg / mL) was mixed with a particle core dispersion having acidic groups on its surface at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol, and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0179] 19-1. Synthesis of Polymer (E202) [Chemistry 23] Polymer Formulation: [Table A-13] Sample Mw. concentration (mM) Final liquid volume (mL) 4-[(2-methyl-1-sideoxy-2-propen-1-yl)amino]butyric acid 171.19 100 1 N-(3-(dimethylamino)propyl)methacrylamide 170.25 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0180] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0181] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred while cooling in an ice bath. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added, and the mixture was stirred overnight. After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was then redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum.

[0182] Dissolve 10 mg of the polymer prepared in the previous step and 28 μL of TEA in DMF (1 mL) and stir. Add 3 mg of the synthetic peptide (with a structure in which the C-terminal carboxyl group is acetylated, having 6 histidines bound from the N-terminus, 3 glycines bound, and lysine bound at the C-terminus) and stir overnight. After the reaction, add a small amount of CH2Cl2, followed by the addition of excess n-hexane to produce a precipitate. Recover the precipitate, add a small amount of CH2Cl2, then add excess n-hexane to wash the precipitate, and then dry it under vacuum.

[0183] The polymer prepared in the preceding step was dissolved in MeOH, and Ni-encapsulated N2,N2-bis(carboxymethyl)-N6-(2-methyl-1-sideoxy-2-propen-1-yl)-L-lysine was added and stirred. CH2Cl2 was added to precipitate the polymer, and the precipitate was recovered and dried under vacuum to obtain polymer (E202).

[0184] 19-2. Formation and Immobilization of Polymer (E202)-Particle Core Complex on Substrate: An aqueous solution of polymer (E202) (2 mg / mL) was mixed with a particle core dispersion having acidic groups on its surface at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol, and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0185] 20-1. Synthesis of Polymer (E203) [Chemistry 24] Polymer Formulation: [Table A-14] Sample Mw. concentration (mM) Final liquid volume (mL) N-(3-aminopropyl)methacrylamide hydrochloride 178.66 100 1 N-(3-(dimethylamino)propyl)methacrylamide 170.25 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0186] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0187] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 40 mg (0.2 mmol) of 4-methylacryloxybenzoic acid was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E₂O₃).

[0188] 20-2. Formation and Immobilization of Polymer (E203)-Particle Core Complex on Substrate: An aqueous solution of polymer (E203) (2 mg / mL) was mixed with a particle core dispersion having acidic groups on its surface at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol, and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0189] 21-1. Synthesis of Polymer (E204) [Chemistry 25] Polymer Formulation: [Table A-15] Sample Mw. concentration (mM) Final liquid volume (mL) GEMA 292.28 100 1 N-(3-(dimethylamino)propyl)methacrylamide 170.25 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0190] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by washing the precipitate with excess n-hexane, and then vacuum drying was performed. The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred while cooled in an ice bath. 59 mg (0.2 mmol) of 4-(2-methylacrylamide ethylaminomethyl)-3-fluorophenylboronic acid was added, and the mixture was stirred overnight (14 h). After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was then redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E₂O₄).

[0191] 21-2. Formation and Immobilization of Polymer (E204)-Particle Core Complex on Substrate: An aqueous solution of polymer (E204) (2 mg / mL) was mixed with a particle core dispersion having acidic groups on its surface at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol, and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain a particle-immobilized substrate.

[0192] 22-1. Synthesis of Polymer (E205) [Chemistry 26] Polymer Formulation: [Table A-16] Sample Mw. concentration (mM) Final liquid volume (mL) 4-[(2-methyl-1-sideoxy-2-propen-1-yl)amino]butyric acid 171.19 100 1 N-(3-(dimethylamino)propyl)methacrylamide 170.25 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0193] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0194] The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred while cooling in an ice bath. 40 mg (0.2 mmol) of EDC and 22 mg (0.2 mmol) of NHS were added, and the mixture was stirred overnight. After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was then redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum.

[0195] 10 mg of the polymer prepared in the previous step was dissolved in 50 mM carbonate buffer (pH 8.5) (1 mL) and stirred. 3 mg of avidin was added and stirred overnight. The reaction solution was placed in a 100 kDa dialysis membrane and dialyzed in phosphate buffer (pH 7.4). After dialysis, the solution was concentrated by ultrafiltration. Subsequently, it was bonded to the biotin monomer ((3aS,4S,6aR)-hexahydro-2-sideoxy-N-[3-[(1-sideoxy-2-propen-1-yl)amino]propyl]-1H-thieno[3,4-d]imidazol-4-pentazolidinylamine) to obtain the target polymer (E205).

[0196] 22-2. Formation and Immobilization of Polymer (E205)-Particle Core Complex on Substrate: An aqueous solution of polymer (E205) (2 mg / mL) was mixed with a particle core dispersion having acidic groups on its surface at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol, and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain the particle-immobilized substrate.

[0197] 23-1. Synthesis of Polymer (E206) [Chemistry 27] Polymer Formulation: [Table A-17] Sample Mw. concentration (mM) Final liquid volume (mL) N-(3-aminopropyl)methacrylamide hydrochloride 178.66 100 1 N-(3-(dimethylamino)propyl)methacrylamide 170.25 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0198] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum. The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred while cooled in an ice bath. Add 38 mg (0.2 mmol) of EDC and 73 mg (0.2 mmol) of 4-[4-[1-(methacryloxy)ethyl]-2-methoxy-5-nitrophenoxy]butyric acid, and stir overnight under light protection. After the reaction, add n-hexane to the reaction solution to recover the precipitate. Dissolve the precipitate again in a small amount of CH₂Cl₂, add excess n-hexane to form a precipitate, recover the precipitate, and dry it under vacuum to obtain the polymer (E₂O₆).

[0199] 23-2. Formation and Immobilization of Polymer (E206)-Particle Core Complex on Substrate: An aqueous solution of polymer (E206) (2 mg / mL) was mixed with a particle core dispersion having acidic groups on its surface at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol, and allowed to stand for 1 hour under light protection. Subsequently, the substrate was washed with EtOH to obtain the particle-immobilized substrate.

[0200] 24-1. Synthesis of Polymer (E207) [Chemistry 28] Polymer Formulation: [Table A-18] Sample Mw. concentration (mM) Final liquid volume (mL) N-(3-aminopropyl)methacrylamide hydrochloride 178.66 100 1 N-(3-(dimethylamino)propyl)methacrylamide 170.25 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0201] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by washing the precipitate with excess n-hexane, and then vacuum drying was performed. The polymer prepared in the previous step and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 38 mg (0.2 mmol) of EDC and 92 mg (0.2 mmol) of compound A (see figure below) were added, and the mixture was stirred overnight under light protection. After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was then redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum to obtain the polymer (E₂O₇). [Chem. 29]

[0202] 24-2. Formation and Immobilization of Polymer (E207)-Particle Core Complex on Substrate: An aqueous solution of polymer (E207) (2 mg / mL) was mixed with a particle core dispersion having acidic groups on its surface at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol, and allowed to stand for 1 hour under light protection. Subsequently, the substrate was washed with EtOH to obtain the particle-immobilized substrate.

[0203] 25-1. Synthesis of polymers (E301-E312) [Chemical Engineering 30] Polymer formulation: [Table A-19] Sample Mw. concentration (mM) Final liquid volume (mL) N-(3-aminopropyl)methacrylamide hydrochloride 178.66 100 1 N-(t-BOC-aminopropyl)methacrylamide 242.31 100 1 NIPAm 113.16 300 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 1 AIBN 164.21 6.25 1

[0204] The compounds in the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, the RAFT agent and initiator were added, and after adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) for 20 h. After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. A small amount of CH₂Cl₂ was added to the residue, followed by the addition of excess n-hexane to wash away the precipitate, and then the residue was dried under vacuum.

[0205] The prepared polymer and 140 μL (1.0 mmol) of TEA were dissolved in CH₂Cl₂ and stirred under ice bath cooling. 36 mg (0.2 mmol) of N-butadiene imine methacrylate was added, and the mixture was stirred overnight. After the reaction, hexane was added to the reaction solution to recover the precipitate. The precipitate was then redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and dried under vacuum.

[0206] Dissolve the polymer in CH₂Cl₂ and stir while cooling in an ice bath. Add 1 mL of dialkyl containing 4 N HCl and stir overnight. Add n-hexane to the reaction solution to recover the precipitate. Add a small amount of CH₂Cl₂ to the precipitate, then add excess n-hexane to wash the precipitate, and then dry under vacuum.

[0207] Dissolve the polymer and 140 μL (1.0 mmol) of TEA in CH₂Cl₂ and stir while cooling in an ice bath. Add 0.25 mmol of any compound from the table on the right and 20 mg (0.1 mmol) of *EDC, and stir overnight. After the reaction, add n-hexane to the reaction solution to recover the precipitate. Redissolve the precipitate in a small amount of CH₂Cl₂, add excess n-hexane to form a precipitate, recover the precipitate and dry it under vacuum to obtain the target polymers (E301-E312). *Use [Table A-20] for the synthesis of E304-E309 and E311-E312. Imported functional groups Compound Examples E301 carboxyl E302 carboxylic acid Active ester group E303 Metal complex (NTA group) E304 Thiol group E305 Pyridine dithio E306 Maleimine E307 Halogenated acetamino groups E308 Azide E309 alkyne group E310 cis-diol group (sugar group) E311 dihydroxyboronic E312 Thiolactone

[0208] 25-2. Formation and Immobilization of Polymer (E301)-Particle Core Complex on Substrate: An aqueous solution of polymer (E301) (2 mg / mL) was mixed with a particle core dispersion having acidic groups on its surface at a 1 / 1 (v / v) ratio. The solution was diluted 80 times with DMF. 4 μL of the solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol, and allowed to stand for 1 hour. Subsequently, the substrate was washed with EtOH to obtain the particle-immobilized substrate.

[0209] (Examples 1-2: Polymer bonding on particles (non-covalent bonds)) 1-2-1. Synthesis of E3-coupled silicon oxide NPs

[0210] [Chemistry 31]

[0211] 100 μL of silicon oxide nanoparticle dispersion (2.0 × 10¹² particles / mL), 500 μL of polymer E3 aqueous solution (2.0 mg / mL), and 400 μL of 4-(4,6-dimethoxy-1,3,5-tris(2-yl)-4-methylmorpholine hydrochloride (DMT-MM) aqueous solution (12.5 mM) were mixed and stirred for 12 hours at 25 °C and 1000 rpm using a Thermo shaker. After the reaction, the reactants in the solution were removed by centrifugation (25 °C, 10,000 g, 15 min) × 5. The supernatants were confirmed by UV-vis measurement (Nano Drop).

[0212] In the same order, polymer E2, polymer E4 and silicon oxide particles are integrated.

[0213] (Results) 1-2-2. DLS and Z-potential determination: 20 μL of silicon oxide nanoparticle dispersion (2.0 × 10¹² particles / mL), 100 μL of aqueous solutions of various cationic polymers (2.0 mg / mL), and 80 μL of pure water were mixed and stirred thoroughly using a vortex mixer. The solution was diluted 100 times with pure water and used as the sample for DLS and Z-potential determination. The determination instrument used was a Zetasizer pro (Marvern Panalytical, UK).

[0214] The particle size measurement results (Figure 1-4 and Table 1-5) and Z-potential measurement results (Figure 1-5 and Table 1-5) obtained by DLS show that the prepared cationic polymer adsorbs onto the silicon oxide nanoparticles in a particulate form, changing from a negatively charged state to a positively charged state. Furthermore, no significant change in particle size was observed before and after the polymer adsorbed onto the silicon oxide nanoparticles, indicating that the silicon oxide nanoparticles maintain monodispersity after polymer adsorption.

[0215] [Table 1-5] Table 1-5 DLS and Z-potential measurements for each particle Average particle size (nm) Surface charge (mV) Silicon oxide nanoparticles 208 -53.2 Silicon oxide nanoparticles + E2 216 56.2 Silicon oxide nanoparticles + E4 237 60.9 Silicon oxide nanoparticles + E3 220 40.5

[0216] (Examples 1-3: Polymer Bonding to Particles) Synthesis of Polymer (E2-0)-Silica Nanoparticle Complex (RM) [Chemical 32] Silicon oxide nanoparticles (200 nm, terminal COOH) at 4.0 × 10¹¹ particles / mL, E2-0 at 2.0 mg / mL, and DMT-MM at 10 mM were mixed in pure water (500 μL) and shaken using a Thermo shaker at 25°C, 1,000 rpm, for 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 min), removing 900 μL of the supernatant. This operation was repeated 5 times to purify the particles. The particle size and surface charge of the obtained particles were measured by DLS, and the results are shown in Figures 1-6. The average particle size (Z-average value) is 232 nm (pdi: 0.022) and the surface charge (Z-potential) is +55.3 mV, thus maintaining the monodispersity of the particles. Furthermore, the surface charge changes significantly to a positive value, confirming that the polymer has been modified.

[0217] (Example 2: Measurement of substrate preparation)

[0218] [Chemistry 33]

[0219] 2-1. Experiment 2-1-1. Surface Modification of Gold Substrate After rinsing the gold substrate (9.8 mm × 4.3 mm) with EtOH, the surface was treated with UV-O3 for 15 min to clean the substrate surface. A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol in EtOH solvent at a ratio of 1:1. The gold substrate was then immersed in this solution. After standing at 30°C for 20 hours, the substrate was washed with EtOH and pure water, thereby forming a self-assembly monolayer (SAM) on the surface of the gold substrate. 2-1-2. Preparation of Particle Immobilized Substrate A silicon oxide nanoparticle dispersion (particle size 200 nm, particle concentration: 2.0 × 10¹¹ particles / mL) and polymer E2 (1.0 mg / mL) were mixed in water at the above final concentration to form a composite.

[0220] The above composite solution was diluted with DMF and added dropwise (4 μL) in the manner shown in Table 2-2, with the number of additions corresponding to the area of ​​the substrate to be immobilized. The solution was then allowed to stand at 25°C for 1 hour. The substrate was then washed sequentially with DMF, EtOH, and pure water to obtain the particle-immobilized substrate.

[0221] The evaluation of the substrate was performed using a fluorescence microscope (KEYENCE, BZ-800), and the analysis was performed using software (hybrid cell counting). The measurement conditions are shown in Table 2-1.

[0222] [Table 2-1] Table 2-1 Experimental Conditions No. Number [each 4 μL] Fluorescence microscopy measurement conditions (exposure time) 4x objective lens 22.2x objective lens 60x objective lens 1 1×10 7 1.5 sec 0.25 sec 0.02 sec 2 3×10 6 ↑ ↑ ↑ 3 1×10 6 ↑ ↑ ↑ 4 3×10 5 2 sec ↑ ↑ 5 1×10 5 ↑ 0.5 sec ↑

[0223] 2-1-3. Synthesis of silicon oxide nanoparticle imprinted polymer on particle-immobilized substrate: A prepolymer solution was prepared according to the composition shown in Table 2-2. After removing dissolved oxygen by cryogenic degassing, polymerization was carried out at 25°C for 18 hours in a glove box. The polymerized substrate was then washed with pure water.

[0224] The above-mentioned substrate was immersed in a 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (methanol: pure water = 1:1) and subjected to ultrasonic treatment for 5 min to remove silicon oxide nanoparticles, thereby forming pores on the surface of the polymer film.

[0225] [Table 2-2] Table 2-2 Composition of Prepolymer Solution Sample concentration main raw materials MPC (2-Methacryloxyethylphosphocholine) 500 mM Initiator EBIB (ethyl α-bromoisobutyrate) 1.25 mM ligands TPMA (tris(2-pyridylmethyl)amine) 0.125 mM catalyst CuBr2 0.0125 mM reducing agent ascorbic acid 1.25 mM solvent EtOH 270 μL

[0226] 2-2. Results 2-2-1. Fabrication of Particle Immobilization Substrate The observation results obtained using fluorescence microscopy are shown in Figure 2-1. Under all conditions, fluorescence from silicon oxide nanoparticles was successfully confirmed on the substrate, thus successfully confirming the immobilization of silicon oxide nanoparticles (polymer complex) on the substrate. Furthermore, the results of fluorescence observation and hybridization cell counting using a 60x objective lens are shown in Figure 2-2. According to Figure 2-2, there is a tendency for the number of silicon oxide nanoparticles on the substrate to increase with the increase of the number of drops. Furthermore, in Figures No. 4 and No. 5, which can be clearly identified as monodisperse, non-aggregated, and monolayered based on the images, there are almost no bright spots with an area exceeding 2 μm², therefore, bright spots with an area exceeding 2 μm² can be identified as aggregates. By using this value as a boundary value, when No.4 (3×10⁶ particles / 4 μL) or more of the bright spots exceeding the boundary value of 2 μm² are produced, aggregation occurs, and when No.5 (1×10⁶ particles / 4 μL) or less, aggregation does not occur. Therefore, it can be objectively represented in numerical form. Furthermore, the particle pattern on the substrate can be controlled by the number of drops applied to the substrate.

[0227] (Example 3: Determination of substrate preparation (cationic polymer)) 3. Study using silicon oxide particles (influence of cationic polymer composition) [Chemical 34]

[0228] 3-1. Experiment 3-1-1. Surface modification of gold substrate After rinsing the gold substrate (9.8 mm × 4.3 mm) with EtOH, UV-O3 treatment was carried out for 15 min to purify the surface of the substrate.

[0229] A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol 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 pure water, thereby forming a self-assembly monolayer (SAM) on the surface of the gold substrate. 3-1-2. Preparation of Particle Immobilization Substrate A silicon oxide nanoparticle dispersion (particle size 200 nm, particle concentration: 2.0 × 10¹¹ particles / mL) and polymers E4 and E3 (1.0 mg / mL) were mixed in water at the above final concentrations to form a composite.

[0230] The above composite solution was diluted with DMF and the solution (4 μL) was dropped onto the substrate in a manner that is relative to the area of ​​the substrate to which the composite is to be immobilized as shown in Table 3-1 or Table 3-2. The solution was then allowed to stand at 25°C for 1 hour.

[0231] The substrate is washed with DMF, EtOH and pure water in sequence to obtain a particle immobilized substrate.

[0232] The evaluation of the substrate was performed using a fluorescence microscope (KEYENCE, BZ-800), and the analysis was performed using software (hybrid cell counting). The measurement conditions are shown in Tables 3-1 and 3-2.

[0233] [Table 3-1] Table 3-1 Experimental Conditions (Polymer E4) No. Number [each 4 μL] Fluorescence microscopy measurement conditions (exposure time) 4x objective lens 22.2x objective lens 60x objective lens 1 1×10 7 1.5 sec 0.25 sec 0.02 sec 2 3×10 6 ↑ ↑ ↑ 3 1×10 6 ↑ ↑ ↑ 4 3×10 5 2 sec ↑ ↑ 5 1×10 5 ↑ 0.5 sec ↑

[0234] [Table 3-2] Table 3-2 Experimental Conditions (Polymer E3) No. Number [each 4 μL] Fluorescence microscopy measurement conditions (exposure time) 4x objective lens 22.2x objective lens 60x objective lens 1 1×10 7 1.5 sec 0.25 sec 0.02 sec 2 3×10 6 ↑ ↑ ↑ 3 1×10 6 ↑ ↑ ↑ 4 3×10 5 ↑ 0.5 sec ↑ 5 1×10 5 ↑ ↑ ↑

[0235] 3-2. Results 3-2-1. Fabrication of Particle Immobilization Substrate The observation results obtained using fluorescence microscopy are shown in Figures 3-1 and 3-2. According to the results, even with different polymer compositions, the particle morphology on the substrate can be controlled by the number of droplets applied to the substrate.

[0236] (Example 4: Measurement of substrate fabrication (glass substrate)) 4. Study using silicon oxide particles (effect on substrate)

[0237] [Chemical 35]

[0238] 4-1-1. Surface finishing of glass substrate: After rinsing the gold substrate (9.8 mm × 4.3 mm) with EtOH, UV-O3 treatment was performed for 15 min to clean the surface of the substrate.

[0239] 3-Aminopropyltriethoxysilane (ATPES) and 3-(trimethoxysilyl)propyl 2-bromo-2-methylpropionic acid were mixed at 1 vol% in EtOH solvent (1% pure water), and the above glass substrate was immersed in the solution. After standing at 25°C for 1 hour, it was washed with EtOH and dried in an oven at 90°C for 2 hours, thereby introducing amino groups and ATRP initiators onto the surface of the glass substrate.

[0240] Prepare a 5 mg / mL succinic anhydride solution (solvent = THF: TEA = 95: 5), and immerse the above-mentioned substrate in it. After standing at 25°C for 4 hours, wash with pure water to change the amine groups on the surface of the glass substrate to carboxyl groups.

[0241] The substrate was immersed in a 1 mM solution of 2-bromoisobutyric acid N-hydroxybutyric acid diimide ester (solvent = DMF), reacted at 25°C for 1 hour, and then washed with EtOH. This yielded a glass substrate with ATRP initiating groups and carboxyl groups on its surface. 4-1-2. Preparation of Particle Immobilized Substrate A silicon oxide nanoparticle dispersion (particle size 200 nm, particle concentration: 2.0 × 10¹¹ particles / mL) and polymer E2 (1.0 mg / mL) were mixed in water at the aforementioned final concentrations to form a composite.

[0242] The above composite solution was diluted with DMF and the solution (4 μL) was dropped onto the substrate in a manner that is relative to the area of ​​the substrate to which the composite is to be immobilized as shown in Table 4-1. The solution was then allowed to stand at 25°C for 1 hour.

[0243] The substrate is washed with DMF, EtOH and pure water in sequence to obtain a particle immobilized substrate.

[0244] The evaluation of the substrate was performed using a fluorescence microscope (KEYENCE, BZ-800), and the analysis was performed using software (hybrid cell counting). The measurement conditions are shown in Table 4-1. Table 4-1 Experimental Conditions No. Number [each 4 μL] Fluorescence microscopy measurement conditions (exposure time) 60x objective lens 1 3×10 7 ? 2 1×10 7 ↑ 3 3×106 ↑ 4 1×10 6 ↑ 5 3×10 5 ↑ 6 1×10 5 ↑ 7 3×10 4 ↑ 8 1×10 4 ↑

[0245] 4-2. Results 4-2-1. Fabrication of Particle Immobilization Substrate The observation results obtained using fluorescence microscopy are shown in Figure 4. According to the results, even with different substrates, the particle morphology on the substrate can be controlled by the number of droplets applied to the substrate.

[0246] (Example 5: Determination of substrate preparation (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, UV-O3 treatment was performed for 15 min to purify the substrate surface.

[0247] A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol in EtOH solvent at a ratio of 1:1. The gold substrate was then immersed in this solution. After standing at 30°C for 20 hours, the substrate was washed with EtOH and pure water, thereby forming a self-assembly monolayer (SAM) on the surface of the gold substrate. 5-1-2. Preparation of particle immobilized substrate: A polystyrene particle dispersion (particle size 250 nm, particle concentration: 2.0 × 10¹¹ particles / mL) and polymer E2 (1.0 mg / mL) were mixed in water at the above final concentration to form a composite.

[0248] The above composite solution was diluted with DMF and added dropwise (4 μL) in a manner that is relative to the area of ​​the substrate to be immobilized as shown in Table 5-1. The solution was then allowed to stand at 25°C for 1 hour.

[0249] The substrate is washed sequentially with DMF, EtOH and pure water to obtain a particle immobilized substrate.

[0250] The evaluation of the substrate was performed using a fluorescence microscope (KEYENCE, BZ-800), and the analysis was performed using software (hybrid cell counting). The measurement conditions are shown in Table 5-1.

[0251] [Table 5-1] Table 5-1 Experimental Conditions No. Number [each 4 μL] Fluorescence microscopy measurement conditions (exposure time) 4x objective lens 22.2x objective lens 60x objective lens 1 1×10 8 0.2 sec 0.05 sec - 2 3×10 7 ↑ ↑ - 3 1×10 7 ↑ ↑ 0.005 sec 4 3×10 6 1 sec 0.2 sec ↑ 5 1×10 6 ↑ ↑ ↑ 6 3×10 5 ↑ ↑ ↑ 7 1×10 5 ↑ ↑ ↑ 8 3×10 4 ↑ ↑ - 9 1×10 4 ↑ ↑ -

[0252] 5-2. Results 5-2-1. Fabrication of Particle-Immobilized Substrate The observation results obtained using fluorescence microscopy are shown in Figures 5-1 and 5-2. Under both conditions, fluorescence from polystyrene particles was successfully confirmed on the substrate, thus successfully confirming the immobilization of polystyrene particles (polymer complex) on the substrate. Furthermore, the obtained fluorescence microscopy images were analyzed using software (hybrid cell counting), and the results are shown in Table 5-2. According to the analysis results, there is a tendency for the number of polystyrene particles on the substrate to increase with the increase of the number of added particles. Especially under the conditions of more added particles (No.1, No.2), the number of bright spots is less than that of No.3, thus the number of aggregates increases. Furthermore, in No. 1 (1×10⁸ particles / 4 μL) and No. 2 (3×10⁷ particles / 4 μL), the particle aggregation on the substrate was relatively large, lacking uniformity. On the other hand, in No. 8 (3×10⁴ particles / 4 μL) and No. 9 (1×10⁴ particles / 4 μL), the amount of immobilized polystyrene particles was extremely small. Therefore, in order to conduct a detailed study on No. 3 (1×10⁷ particles / 4 μL) to No. 7 (1×10⁵ particles / 4 μL), fluorescence observation was performed using a 60x objective lens, and hybridization cell counting was used for analysis. The results are shown in Figures 5-3 and 5-4, respectively. According to Figure 5-3, as the number of drops increases, the area of ​​the largest bright spot increases, and the aggregation scale becomes larger. Furthermore, in No. 6 and No. 7, which can be clearly identified as monodisperse, non-agglomerated, and monolayered based on the images, the area of ​​the bright spots is approximately 2 μm². Therefore, bright spots with an area exceeding 2 μm² can be identified as agglomerated particles. Using this value as a boundary value, the number of bright spots above the boundary values ​​of 2 μm², 4 μm², and 10 μm² is shown in Figure 5-4. According to this result, when No. 4 (3 × 10⁶ particles / 4 μL) or more bright spots exceeding the boundary value of 2 μm² are generated, agglomeration occurs; when No. 5 (1 × 10⁶ particles / 4 μL) or less, no agglomeration occurs. Therefore, the particle morphology on the substrate can be controlled based on the number of drops applied to the substrate.

[0253] [Table 5-2] Table 5-2 Analysis of hybrid cell counts using images taken with a 22.2x objective lens Added concentration Total number of highlights Number of single-dispersion bright spots (≤10 μm) 2 ) Number of bright spots in condensed aggregates (>10 μm) 2 ) Maximum bright spot area [μm] 2 ] Total bright spot area [μm] 2 ] 1×10 8 4059 3716 343 454.911 12072.21 3×10 7 9152 7648 1504 223 29613 1×10 7 16441 14889 1552 423 33967 3×10 6 8472 7364 1108 150 20317 1×10 6 4289 3377 912 39.029 20715.15 3×10 5 258 181 77 57 1145 1×10 5 61 37 twenty four 20.412 191.914 3×104 30 26 4 15 89 1×10 4 12 9 3 twenty two 66

[0254] (Example 6: Performance Test (Evaluation of Particle Density on Substrate)) Study on Recess Density on 6A Substrate Using Fluorescent Cationic Polymer

[0255] [Chemistry 36]

[0256] 6-1. Experiment 6-1-1. Surface modification of gold substrate After rinsing the gold substrate (9.8 mm × 4.3 mm) with EtOH, UV-O3 treatment was carried out for 15 min to purify the surface of the substrate.

[0257] A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyroxy)undecylthiol and carboxyl-EG6undecylthiol in EtOH solvent at a ratio of 1:1. The gold substrate was then immersed in this solution. After standing at 30°C for 20 hours, the substrate was washed with EtOH and pure water, thereby forming a self-assembly monolayer (SAM) on the surface of the gold substrate. 6-1-2. Preparation of Particle Immobilization Substrate A silicon oxide nanoparticle dispersion (particle size 200 nm, particle concentration: 2.0 × 10¹¹ particles / mL) and polymer F1 (1.0 mg / mL) were mixed in water at the above final concentration to form a composite.

[0258] The above composite solution was diluted with DMF and added dropwise (4 μL) in a manner that is relative to the area of ​​the substrate to be immobilized as shown in Table 6-1, and then allowed to stand at 25°C for 1 hour.

[0259] The substrate is washed with DMF, EtOH and pure water in sequence to obtain a particle immobilized substrate.

[0260] The evaluation of the substrate was performed using a fluorescence microscope (KEYENCE, BZ-800), and the analysis was performed using software (hybrid cell counting). The measurement conditions are shown in Table 6-1.

[0261] [Table 6-1] Table 6-1 Experimental Conditions (Polymer FI) No. Number [each 4 μL] Fluorescence microscopy measurement conditions (exposure time) green red After particle immobilization After aggregation After template removal After particle immobilization After aggregation After template removal 1 1×10 7 0.1 sec 0.1 sec 0.2 sec 0.01 sec 0.01 sec 0.02 sec 2 3×10 6 ↑ ↑ ↑ ↑ ↑ ↑ 3 1×10 6 ↑ ↑ 0.5 sec ↑ ↑ 0.05 sec

[0262] 6-1-3. Synthesis of silicon oxide nanoparticle imprinted polymer on particle-immobilized substrate. A prepolymer solution with the composition shown in Table 6-2 was prepared. After removing dissolved oxygen by cryogenic degassing, polymerization was carried out at 25°C for 18 hours in a glove box. The polymerized substrate was then washed with pure water.

[0263] The above-mentioned substrate was immersed in a methanol:pure water = 1:1 solution and subjected to ultrasonic treatment for 0.5 min to remove silicon oxide nanoparticles, thereby forming pores on the surface of the polymer film.

[0264] [Table 6-2] Table 6-2 Composition of Prepolymer Solution material concentration main raw materials MPC (2-Methacryloxyethylphosphocholine) 500 mM Initiator EBIB (ethyl α-bromoisobutyrate) 1.25 mM ligands TPMA (tris(2-pyridylmethyl)amine) 0.125 mM catalyst CuBr2 0.0125 mM reducing agent ascorbic acid 1.25 mM solvent EtOH 270 μL

[0265] 6-2. Results 6-2-1. Fabrication of Particle Immobilization Substrate Regarding the composite of silicon oxide nanoparticles and fluorescent cationic polymer F1, the particle size and surface Z-potential were measured using a Zeta Sizer, and the results are shown in Table 6-3. Before and after the formation of the composite, the surface Z-potential changed from a negative value to a positive value, thus successfully confirming that the silicon oxide nanoparticles and fluorescent cationic polymer F1 formed a composite. Furthermore, based on the particle size and PDI, it was successfully confirmed that the particles did not agglomerate and their dispersibility was not compromised.

[0266] [Table 6-3] Table 6-3 Results of particle size and surface Z-potential measurements using Zeta Sizer Analyte Particle size PDI Surface Z potential Silicon oxide nanoparticles 201.2 nm 0.01824 -29.97mV Silicon oxide nanoparticles + F1 complex 225.8 nm 0.1005 57.94mV

[0267] Furthermore, the observation results obtained using fluorescence microscopy are shown in Figure 6-1. Green fluorescence from silicon oxide nanoparticles and red fluorescence from fluorescent cationic polymers were successfully confirmed in any sample, thus confirming that the silicon oxide nanoparticle-fluorescent cationic polymer composite was fixed on the substrate.

[0268] 6-2-2. Fluorescent cationic polymer F1 after removal of silicon oxide nanoparticles. The observation results obtained by fluorescence microscopy after polymerization of the polymer layer on the substrate after immobilization of silicon oxide nanoparticle-fluorescent cationic polymer composite, and the observation results obtained by fluorescence microscopy after removal of silicon oxide nanoparticles after polymerization are shown in Figures 6-2 and 6-3, respectively. According to Figure 6-2, fluorescence from silicon oxide nanoparticles and fluorescent cationic polymer was successfully confirmed on the substrate after polymerization. However, according to Figure 6-3 after removal of silicon oxide nanoparticles, green fluorescence from silicon oxide nanoparticles could not be confirmed, but red fluorescence from fluorescent cationic polymer was successfully confirmed. It is thus confirmed that after removing silicon oxide nanoparticles through experimental operation, fluorescent cationic polymer still remains in the polymer layer (within the pores after removal of silicon oxide nanoparticles) made on the substrate.

[0269] Furthermore, the red fluorescence remaining on the substrate after the removal of silicon oxide particles originates from the cationic polymer present in the pores after the removal of silicon oxide particles. Therefore, the pore density on the substrate is calculated by equating the number of bright spots of this red fluorescence with the number of pores. The results show that, under the experimental conditions, the pore density formed on the substrate is related to the number of silicon oxide particles dropped onto the substrate, ranging from 10³ to 10⁶ particles / mm². Therefore, it is known that by controlling the number of particles dropped onto the substrate, not only can the particle morphology on the substrate be controlled, but the pore density after the removal of silicon oxide particles can also be controlled.

[0270] 6. B. Particle density on substrate: Based on the results of analyzing the particles on the substrates obtained in Examples 2, 3, and 5 using fluorescence observation with a 60x objective lens and hybridization cell counting (Figures 3-1, 3-2, and 5-1), it can be seen that the number of silicon oxide nanoparticles on the substrate tends to increase with the increase of the number of drops. Furthermore, in No. 4 and No. 5, which can be clearly identified as monodisperse, non-agglomerated, and monolayered based on the images, almost no bright spots have an area exceeding 2 μm². Therefore, bright spots with an area exceeding 2 μm² can be identified as agglomerated aggregates. By using this value as a boundary value, aggregation occurred when the number of bright spots exceeding 2 μm² (No. 4, 3 × 10⁶ particles / 4 μL) exceeded the boundary value, while no aggregation occurred when the number of bright spots (No. 5, 1 × 10⁶ particles / 4 μL) was below the boundary value. Therefore, the distribution can be objectively represented numerically, and the particle pattern on the substrate can be controlled based on the number of drops applied to the substrate. The analysis results of the particles present on the measured substrate are summarized in Figure 6-4. When the particles were dispersed at a concentration of less than No. 5 (1 × 10⁶ particles / 4 μL) without aggregation, and when the particles were present on the substrate at a particle density of less than 1 × 10⁵ particles / mm², the particles were monodisperse, non-aggregated, and arranged in a monolayer on the substrate.

[0271] (Example 7: Composite of His-tagged polymer with silicon oxide nanoparticles) [Chemical 37][Table B] Sample Mw. concentration. (mM) Final liquid volume (mL) weight (mg) N-Acryloylhomocysteine ​​Thiolactone 171.22 100 1 17 N-(3-(dimethylamino)propyl)methacrylamide 170.25 200 1 35 NIPAm 113.16 700 1 79 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 10 1 3.5 AIBN 164.21 5 1 0.8 The compounds from the above formulation were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). RAFT agent and initiator were added. After adding a stir bar, the mixture was degassed with argon and polymerized in an oil bath (set temperature: 70 °C) for 17 h (stirring speed 300 rpm). After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. The precipitate was washed with diethyl ether and dried under vacuum. 5 mg of the obtained polymer, 4 mg of the His tag (with a structure in which the C-terminal carboxyl group is acetylated by binding 6 histidines from the N-terminus, 3 glycines, and lysines from the C-terminus), 3.5 mg of pyridyl dithioethyl-PEG3-methylacrylamide, and 2.8 μL of TEA were dissolved in DMF (0.3 mL) and stirred for 9 hours. CH₂Cl₂ and n-hexane were added to the reaction solution, and the resulting precipitate was recovered and dried under vacuum to obtain the polymer (E11His). iii) E11His was added dropwise to a gold substrate modified with a 2-(2-bromoisobutyryloxy)undecylthiol and NTA-SAM formation reagent, followed by standing (room temperature, 15 min), and then the substrate was washed with pure water. A mixed solution of silicon oxide nanoparticles (200 nm, COOH ends) and E11His aqueous solution (silicon oxide concentration 2.0 × 10¹¹ particles / mL, polymer concentration 1 mg / mL) was diluted 4,400 times with DMF. 4 μL of this solution was added dropwise to the gold substrate after the reaction with NiCl₂ and allowed 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.

[0272] (Example 8: Cationic silicon oxide nanoparticles + Anionic polymer Ex8) 1. Synthesis method of anionic polymer Ex8 [Chemical 38] The compounds in the formulation of Table C were placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 1 mL). Finally, RAFT agent and initiator were added, and after adding a stir bar, degassing with argon gas was performed. Polymerization was carried out in an oil bath (set temperature: 70 °C) (16 h). After polymerization, the Schlenk flask was cooled in an ice bath, and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. The recovered precipitate was washed with diethyl ether and then dried under vacuum to obtain the polymer. The obtained polymer and 70 μL (0.5 mmol) of TEA were dissolved in CH2Cl2 and stirred under ice bath cooling. 54 mg (0.3 mmol) of N-butadiene imine methacrylate was added and stirred overnight. Subsequently, hexane was added to the reaction solution to recover the precipitate. The precipitate was then redissolved in a small amount of CH₂Cl₂, and excess hexane was added to produce another precipitate. This precipitate was recovered and vacuum dried to obtain the target polymer (Ex8). Yield: 120 mg [Table C] Sample Mw. concentration. (mM) Final liquid volume (mL) N-(3-aminopropyl)methacrylamide hydrochloride 178.66 100 1 4-[(2-methyl-1-sideoxy-2-propen-1-yl)amino]butyric acid 171.19 200 1 Isopropyl acrylamide (NIPAm) 113.16 700 1 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 10 1 AIBN 164.21 5 1 2. Composite of Silicon Oxide Nanoparticles with Polymers: 1.98 mg (39.6 μL) of silicon oxide (red, flat, 200 nm, 50 mg / mL) was mixed with 60.4 μL of pure water (total 100 μL), and added to 800 μL of EtOH pre-filled in a 1.5 mL microcentrifuge tube. Then, 100 μL of 2 M HCl and 15 μL of APTES were added. The reaction was carried out at 25 °C using a Thermo shaker at 1000 rpm for 20 h. After the reaction, the nanoparticles were washed with ethanol by centrifugation (5000 rpm, 10 min × 3). 20 μL of the prepared silicon oxide nanoparticle dispersion (NH₂-terminated, 200 nm, 4.0 × 10¹¹ particles / mL, 50 mM carbonate buffer (pH 8.0)) was mixed with 20 μL of Ex8 carbonate buffer solution (2 mg / mL) and allowed to stand at 25 °C for 15 minutes. 3. DLS Measurement: The composite of particles 2 was diluted 100-fold with pure water, and the particle size and Z-potential were measured using DLS. 4. Immobilization on Substrate: The composite of particles 2 was diluted 80-fold with DMF. 4 μL was added to a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol:amino-EG6undecylthiol (1:1 vol) and allowed to stand at 25 °C for 1 h. The surface was then washed with EtOH and observed using a fluorescence microscope and SEM (Figure 8). [Chem. 39] Based on the particle size and Z-potential measurements obtained using DLS, it is indicated that the anionic polymer adsorbed onto the silicon oxide nanoparticles changes from a positively charged state to a negatively charged state. Furthermore, the particle size did not change significantly, indicating that the dispersibility was maintained after polymer adsorption. [Table D] Silicon oxide nanoparticles Ex8 composite Particle size (nm) 221.5 251.7 Z-potential (mV) 34.4 -14.4

[0273] (Results) Based on the observations in Figure 8, the fluorescent bright spots from the silicon oxide nanoparticles were observed to be monodisperse, non-aggregated, and distributed in a monolayer. This indicates that even when the anionic polymer Ex8 is combined with cationic silicon oxide nanoparticles, the same situation as with cationic polymer Ex8 and anionic silicon oxide nanoparticles occurs, allowing the particles to be fixed on the substrate in a monodisperse, non-aggregated, and monolayer manner.

[0274] (Example 9: Cationic nanoparticles + anionic polymer) [Chemical 40]

[0275] (Silicon oxide nanoparticles used) • E2-0 coupled red-COOH silicon oxide (RM) 200 nm 4.0×10¹¹ particles / ml

[0276] (Polymer solution) • Final polymer concentration 0.87 mg / mL (10 mM PB, pH 7.47)

[0277] (Anionic Silicon Oxide) • A silicon oxide nanoparticle dispersion was mixed with polymer AN-1 to form a composite. (Final silicon oxide concentration: 1.0 × 10¹¹ particles / ml; final polymer concentration: 0.435 mg / mL (5 mM PB)) The above composite solution was diluted 4, 40, 400, and 4000 times with DMF. 4 μL of the solution was added dropwise to the substrate to which the composite was to be immobilized, and the solution was allowed to stand at 25°C for 1 hour. The substrate was then washed with ethanol to obtain the particle-immobilized substrate.

[0278] (Polymer matrix synthesis) Thin film was formed by ATRP (25°C, 20 h).

[0279] (Particle nucleus removal element) Add 300 μL of 50% acetic acid / MeOH to a 1.5 mL tube, immerse each substrate individually, perform ultrasonic treatment for 8 minutes, and then rinse with pure water.

[0280] (SS reduction and cleavage) Add to 50 mM tris(2-carboxyethyl)phosphonic acid hydrochloride in 50% MeOH aqueous solution and react at 40°C for 1 h. Wash the substrate with pure water after reaction.

[0281] (Introduction of fluorescent dye) 30 μL of a 50 μm solution of Alexa 647-C2-maleimide and a 50 μm solution of maleimide-C3-NTA (5% DMSO 10 mM PB(7.4)) was dropped onto the substrate and allowed to stand at 25°C for 1 h to react. The substrate was then washed with pure water. The solution was installed in a flat pipette tip manufactured by Fukami Chemical, and the change in fluorescence intensity on the substrate surface was measured using an automated dispensing device with an attached fluorescence microscope. Sequence 1. Wafer mounting / PBS aspiration (150 μL), obtaining images before antibody immobilization. 2. Ni complex formation: Aspirate 100 μL of 4 mM NiCl2 (aqueous solution), react for 5 min (25℃), wash with pure water × 4 times, aspirate 150 μL and spray. 3. Protein G immobilization: Aspirate 100 μL of 1 μm His-protein G (PBS, abcam), react for 10 min (25℃), wash with 0.01% Tween (registered trademark) 20 PBS × 2 times, PBS × 2 times, aspirate 150 μL and spray. 4. Antibody immobilization: Aspirate 100 nM anti-CD9 (commercially available) (PBS) 100 μL, react for 10 min (25℃), wash with 0.01% Tween (registered trademark) 20 PBS × 2 times, PBS × 2 times, aspirate 150 μL and spray. 5. Background determination (F0: 0.1% BSA, 100 5 times with 0.15 M PB (pH 7.0), 0.15 M NaCl. 6. Extracellular adsorption adjusted to 0, 3, 30, 300, 1000 fM SKBR3 culture supernatant extracellular ions (0.1% BSA, 100 mM PB (pH 7.0), 0.15 M NaCl). Aspirate 100 μL of sample (extracellular ions from SKBR3), react for 5 min (25℃). Image acquisition: Fluorescence microscope camera: Zyla 4.2; Filter: Cy5 (excitation wavelength 604-644 nm, fluorescence wavelength 672-712 nm); Objective lens: ×5; Exposure time: 0.5 sec; Light intensity: 12%; Light source: mercury lamp. Aspirate 150 μL of 0.01% Tween (trademark) 20 PBS and spray out. Repeat 1-6 times.

[0282] (Results) For the sensing chip made by modifying silicon oxide particles with anionic polymer on a cationic surface substrate, it was successfully confirmed that by adding 10 fM of extracellular material derived from the SK-BR3 cell line, there was a change of about 5% in relative fluorescence intensity.

[0283] (Example 10: Biodegradable nanoparticles: polylactic acid / glycolic acid copolymer (PGLA)) [Chemical 41]

[0284] (SAM Formation on Substrate) A gold substrate was immersed in a 0.5 mM solution of 2-(2-bromoisobutyroxy)undecylthiol and a 0.5 mM solution of carboxyl-EG6undecylthiol (EtOH) to form SAM. (30°C, 20 h)

[0285] (Particle preparation) After 1 min of ultrasonic (Lv2) treatment of the aqueous suspension of fluorescent carboxylated PGLA nanoparticles (200 nm), the nanoparticles and E2 polymer aqueous solution were mixed to a final concentration of 2×10¹¹ particles / mL and 1 mg / mL, respectively. The solution was diluted 80 times with EtOH, and 4 μL of the resulting solution was dropped onto the substrate (1.0×10⁷ particles / 4 μL). After standing at 25 °C for 1 h, the substrate was washed 3 times with EtOH.

[0286] (SEM observation, fluorescence observation) After mixing the E2 polymer aqueous solution to a concentration of 1 mg / mL, it was diluted with EtOH. 4 μL of the resulting solution was dropped onto the substrate (1.0 × 10⁷ particles / 4 μL). After standing at 25°C for 1 h, it was washed three times with EtOH. Fluorescence observation was then performed on the substrate using EtOH (KEYENCE BZ-X800, excitation wavelength 470±20 nm / fluorescence wavelength 525±25 nm). Subsequently, vacuum drying and gold sputtering were performed, followed by scanning electron microscopy (SEM) image observation. (Figure 9)

[0287] (Results) The results showed a state comparable to that of silicon oxide nanoparticles or polystyrene nanoparticles, indicating that the method can be applied to all nuclear particles.

[0288] (Example 11: Silicon oxide nanoparticles + cationic polymer E2P20 containing aromatic components) 1. Synthesis method of E2P20 [Chemical 42] The cationic polymer E2P20 containing aromatic components (phenyl) was synthesized as follows. The compound of the above formulation was placed in a Schlenk flask (25 mL) and dissolved in DMF (final volume: 2 mL). Finally, RAFT agent and initiator were added. After adding a stir bar, the mixture was degassed with argon gas and polymerized in an oil bath (set temperature: 75°C) (24 h). (Stirring speed 300 rpm) After polymerization, the Schlenk flask was cooled in an ice bath and the reaction was stopped by introducing air. The reaction solution was added to a large amount of diethyl ether to recover the precipitate. The precipitate was washed with diethyl ether and dried under vacuum. The polymer prepared in the previous step and 42 μL of TEA were dissolved (or dispersed) in CH2Cl2 and stirred. 37 mg of N-butadiene imine methacrylate was added and stirred overnight. After the reaction, hexane was added to the reaction solution to recover the precipitate. MeOH, CH₂Cl₂, and hexane were added to the precipitate, and the precipitate was recovered. The mixture was then vacuum dried to obtain the target polymer (E₂P₂₀). Yield: 110 mg [Table E] Sample Mw. concentration (mM) Final liquid Volume (mL) weight (mg) Liquid volume (μL) Cys Methacrylamide 256.82 100 2 51.364 - N-(3-(dimethylamino)propyl)methacrylamide 170.25 100 2 34.050 36.223 N-Phenylacetylamine 147.18 100 2 29.436 N-Isopropylacrylamide (NIPAm) 113.16 200 2 45.264 - 2-Cyano-2-[(dodecylthiothiocarbonyl)thio]propane 345.63 12.5 2 8.641 - AIBN 164.21 6.25 2 2.053 -

[0289] 2. Composite of silicon oxide nanoparticles and polymers: 40 μL of aqueous dispersion of silicon oxide nanoparticles (COOH end, 200 nm, Lot.0921940-03, 4.0×10 11 particles / mL) was mixed with 40 μL of aqueous solution of E2P20 (2 mg / mL) and allowed to stand at 25°C for 15 minutes to form a composite of silicon oxide nanoparticles and E2P20.

[0290] 3. DLS determination: The complex of silicon oxide nanoparticles and E2P20 was diluted 100 times with pure water, and the particle size and Z-potential were determined by DLS.

[0291] 4. Immobilization on the substrate: The composite of silicon oxide nanoparticles and E2P20 was diluted 80 times with DMF and 4 μL was dropped onto a gold substrate modified with 2-(2-bromoisobutyroxy)undecylthiol:carboxyl-EG6undecylthiol (1:1). The substrate was then allowed to stand at 25°C for 1 h. The surface was subsequently cleaned with EtOH and observed using fluorescence microscopy and SEM. The particle size and Z-potential measurements obtained using DLS indicated that the cationic polymer adsorbed onto the silicon oxide nanoparticles, changing from a negatively charged state to a positively charged state. Furthermore, the particle size did not change significantly, indicating that the dispersibility was maintained after polymer adsorption. [Table F] Silicon oxide nanoparticles E2P20 composite Particle size (nm) 210.1 223.2 Z-potential (mV) -41.2 45.8

[0292] (Results) Based on the observations shown in Figure 10-1, the fluorescent bright spots from the silicon oxide nanoparticles were observed to be monodisperse, non-aggregated, and distributed in a monolayer. This indicates that E2P20, which incorporates an aromatic component (phenyl) into E2, exhibits the same characteristics as E2. When combined with silicon oxide nanoparticles and fixed onto the substrate, it is monodisperse and non-aggregated, and can be fixed onto the substrate in a monolayer.

[0293] (Example 12: Polystyrene nanoparticles + cationic polymer E2P20 containing aromatic components) Polystyrene nanoparticles were used instead of the silicon oxide nanoparticles in Example 11, and were composited with cationic polymer E2P20 containing aromatic components. [Chemical 43] 20 μL of polystyrene (PS) nanoparticles (250 nm, COOH end, 2.0 × 10¹² particles / mL) were mixed with 20 μL of E2P20 aqueous solution (2 mg / mL), and the solution was diluted with DMF to 40, 400, 4000, and 40000 times. 4 μL of each solution was dropped onto a gold substrate modified with 2-(2-bromoisobutyryloxy)undecylthiol and carboxyl-EG 6undecylthiol, and allowed to stand for 1 hour. The surface was then washed with EtOH and observed under a fluorescence microscope. (Fig. 10-2)

[0294] (Results) Similar to the case with silicon oxide nanoparticles, even when using polystyrene nanoparticles, regardless of the number of particles added, the fluorescent bright spots were monodisperse and unaggregated, distributed in a monolayer. This indicates that even when aromatic components are introduced into cationic polymers, the particles can be fixed in a monodisperse, unaggregated, and monolayer manner.

[0295] (Example 13: Performance Test (Sensor Sensitivity), Reproducibility Confirmation) 13-1. Experiment 13-1-1. Surface Modification of Gold Substrate After cleaning the gold substrate (9.8 mm × 4.3 mm) with EtOH, UV-O3 treatment was performed for 15 min to clean the substrate surface. A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyryloxy)undecylthiol and carboxyl-EG 6undecylthiol in EtOH solvent at a 1:1 ratio. The gold substrate was then immersed in this solution. After standing at 30°C for 20 hours, the substrate was cleaned with EtOH and pure water, thereby forming a self-assembly monolayer (SAM) on the surface of the gold substrate. 13-1-2. Preparation of particle immobilization substrate: A dispersion of silicon oxide nanoparticles in water (terminal functional group: COOH, particle size 200 nm, particle concentration: 4.0 × 10¹¹ particles / mL) was mixed with an aqueous solution of polymer E2 (2.0 mg / mL) at a ratio of 1 / 1 (v / v) to form a composite.

[0296] The above composite solution was diluted with DMF to form 1×10⁸, 1×10⁷, 1×10⁶, and 1×10⁵ particles, and then added dropwise (4 μL) to the substrate to be immobilized. The substrate was allowed to stand at 25°C for 1 hour. The substrate was then washed with DMF and EtOH sequentially to obtain the particle-immobilized substrate. Ten substrates were prepared for each addition condition. 13-1-3. Synthesis of polymer matrix and analytical sensor on particle-immobilized substrate The prepolymer solution was prepared with the composition shown in Table 7. After removing dissolved oxygen by cryogenic degassing, the prepolymer was mixed in an argon-purified glove box and polymerized at 25°C for 20 hours. The polymerized substrate was washed with a large amount of pure water.

[0297] The polymerized substrate was immersed in an AcOH / MeOH (1 / 1 (v / v)) solution and subjected to ultrasonic irradiation for 5 min. The substrate was then washed with MeOH and pure water to remove silicon oxide nanoparticles, thereby forming recesses in the polymer film. Subsequently, the substrate was immersed in a 50 mM tris(2-carboxyethyl)phosphine hydrochloride solution (methanol / pure water = 1 / 1 (v / v)) and subjected to ultrasonic treatment for 5 min to remove the E2 polymer and expose the thiol groups.

[0298] [Table 7] Table 7 Composition of Prepolymer Solution Sample concentration main raw materials MPC (2-Methacryloxyethylphosphocholine) 500 mM Initiator EBIB (ethyl α-bromoisobutyrate) 1.25 mM ligands TPMA (tris(2-pyridylmethyl)amine) 0.125 mM catalyst CuBr2 0.0125 mM reducing agent ascorbic acid 1.25 mM solvent EtOH 270 μL

[0299] A 50 μM Alexa Fluor(R)647 C2 maleimide and a 50 μM maleimide C3 NTA PBS solution (30 μL) were added to the above substrate and allowed to stand at 25°C for 1 hour to introduce fluorescent molecules and NTA groups into the recesses. The substrate was then washed with MeOH and pure water after the reaction.

[0300] 4 mM NiCl2 aqueous solution (30 μL) was added to the above substrate and allowed to stand at 25°C for 30 minutes to form a Ni-NTA complex. The substrate was washed with pure water after the reaction. Subsequently, 1 μM of His-tagged protein G PBS solution (30 μL) was added and allowed to stand at 25°C for 1 hour to introduce protein G by forming a complex between Ni-NTA and the His-tagged protein. Finally, 0.3 μM of anti-CD9 antibody PBS solution (30 μL) was added and allowed to stand at 25°C for 1 hour to construct the analytical sensor. 13-1.4. Adsorption Experiment An extracellular body capture experiment of prostate cancer cells (PC-3) was performed using a fluorescence microscope (manufactured by SYSTEM INSTRUMENTS Inc.) with an automated dispensing device. The concentration of PC-3 extracellular body PBS solution (10 mM phosphate, 140 mM NaCl, pH 7.4) was set to 0 and 1.0 fM. Regarding the measurement conditions for fluorescence microscopy, the filter was Cy5 (excitation wavelength 604-644 nm, fluorescence wavelength 672-712 nm), the objective lens was 5x, the exposure time was 0.2 sec, the light intensity was 12%, and the light source was a mercury lamp. Measurements were performed within the central area of ​​the substrate. The sequence of the automated dispensing device was as follows: 1. wafer mounting, 2. sample aspiration (100 μL), 3. reaction (1 min, 25°C), 4. complete ejection, 5. aspiration of 150 μL of 10 mM PBS (140 mM NaCl, pH 7.4), 6. maintaining the measurement position (repeated 2→6 below). The initial fluorescence intensity (F0) and the relative fluorescence intensity changes upon extracellular addition were measured for 10 analytical sensors fabricated using substrates with immobilized particle numbers of 1×108, 1×107, 1×106, and 1×105. The coefficient of variation (= standard deviation / average [%)) was calculated based on the average value and standard deviation of the relative fluorescence intensity changes of each analytical sensor. The correlation between the number of immobilized particles and the coefficient of variation of the fabricated analytical sensor was observed to study the effect of the number of immobilized particles on reproducibility.

[0301] (Comparative Example 1: Aggregation and immobilization of silicon oxide nanoparticles under the conditions of International Publication No. 2018 / 221271 and Toshifumi Takeuchi et al., J. Am. Chem. Soc., March 10, 2020, Vol. 142, Page. 6617-6624) 1. Introduction of His tag and thiol group into silicon oxide nanoparticles Fluorescent silicon oxide nanoparticles (red-COOH, 200 nm, Lot. 0442240-01) were adjusted with water to 25 mg / mL (3.0 × 10 12 particles / mL). 10 μL of a 50 mM His-tagged (a structure with 6 histidine groups bound from the N-terminus, 3 glycine groups bound from the N-terminus, and lysine group bound from the C-terminus, where the C-terminal carboxyl group is acetylated) aqueous solution, 10 μL of a 50 mM 2-aminoethanethiol-HCl aqueous solution, and 10 μL of a 50 mM DMT-MM aqueous solution were added to 1 mL of an aqueous suspension of silicon oxide nanoparticles. The reaction was carried out at 25°C and 1000 rpm for 19 hours using a Thermo shaker, thereby introducing His-tagged and thiol groups into the silicon oxide nanoparticles. 2. Substrate fabrication: A gold substrate was immersed in a 0.5 mM solution of 2-(2-bromoisobutyryloxy)undecylthiol and a 0.5 mM amino-EG6undecylthiol EtOH solution to introduce amino and bromine groups onto the substrate surface (30°C, 20 h). 3. Formation of Ni complex by introducing NTA onto the substrate: Add 80 μL of 5 mM DMSO solution of isothiocyanobenzyl-NTA to the substrate with introduced amino and bromine groups, and let stand at 25°C for 2 hours. Wash twice with DMSO and twice with pure water, then dry with N2. Add 30 μL of 4 mM NiCl2 aqueous solution, let stand at 25°C for 15 minutes, and wash three times with pure water to form the Ni complex of NTA. 4. Immobilization of His-tagged and thiol-group-containing silicon oxide nanoparticles: Add 50 μL of phosphate-buffered saline (PBS) to the above substrate at the concentrations shown in the table, and let stand at 25°C for 1 hour. Wash the substrate three times with PBS, and observe the substrate surface using a fluorescence microscope (Keyence BZ-800) and SEM. (Figure 11) [Table G] operate Particle concentration (mg / mL) Solvent types Dropping volume (μL) Number of droplets 1.0-PBS 1.0 PBS 50 6.0×10 9 Particles / 50 μL 5.1-PBS 5.1 PBS 50 3.1×10 10 Particles / 50 μL Based on the observations in Figure 11, individual silicon oxide particles were observed in some areas, but most of them were observed to be aggregated. This indicates that when silicon oxide nanoparticles were modified with His tags and then fixed onto the substrate, they could not be monodispersed and aggregated, and were not immobilized in a monolayer.

[0302] (Comparative Example 2: Sensitivity of a sensor fabricated without the use of particles) [Chemical 44] (Formation of SAM on a substrate) A gold substrate (9.8 mm × 4.3 mm) was treated with UV-O3 for 20 min to clean the substrate surface. A 1 mM EtOH solution was prepared by mixing 2-(2-bromoisobutyroxy)undecyl mercaptan and carboxyl-EG6undecyl mercaptan in EtOH solvent at a ratio of 1:1. The gold substrate was then immersed in the solution. After standing at 30°C for 20 hours, the substrate was washed with EtOH, thereby forming a self-assembly monolayer (SAM) on the surface of the gold substrate. An aqueous solution of polymer E2 (1 mg / mL) was diluted 80 times with DMF, and 4 μL was added to the substrate with the SAM formed. The substrate was then stood at 25°C for 1 hour. The substrate was then washed with ethanol.

[0303] (Polymer matrix synthesis) A prepolymer solution and ascorbic acid solution were prepared, dissolved oxygen was removed by cryogenic degassing, and then mixed in a glove box. Polymerization was carried out at 25°C for 20 hours. The polymerized substrate was then washed with pure water. [Table H] ATRP formulation MPC 500 mM ethyl α-bromoisobutyrate 1.25 mM TPMA 0.125 mM CuBr2 0.0125 mM L-Ascorbic Acid 1.25 mM EtOH 270 μL

[0304] (Acetic acid treatment) Place in a 50% MeOH-50% acetic acid solution, perform ultrasonic treatment for 5 min, and then rinse with pure water.

[0305] (SS Reduction Cutting) Add 0.5 mM tris(2-carboxyethyl)phosphonic acid hydrochloride aqueous solution to a Thermo shaker, preheat at 40°C for 5 min, then place the acetic acid-treated substrate in the shaker and react at 40°C for 1 min. Wash the reacted substrate with pure water.

[0306] (Fluorescence, NTA introduction) Add 30 μL of a 10 mM PB (pH 7.4) solution containing 50 μm maleimide-C3-NTA-50 μm Alexa 647-C2-maleimide in 20% DMSO, and let it stand at 25°C for 1 h to react. Then wash with DMSO and pure water.

[0307] (His-tagged protein G, antibody introduction) 30 μL of 4 mM NiCl2 aqueous solution was dropped onto the substrate and allowed to stand (25°C, 15 min). The substrate was washed with pure water after the reaction. 30 μL of 1 μm His-tagged protein G (His-Protein G) PBS solution was added and allowed to stand at 25°C for 1 hour to immobilize His-Protein G. The His-Protein G immobilized substrate was washed with PBS. 30 μL of 0.1 μm anti-CD9 PBS solution was added and allowed to stand at 25°C for 1 hour to introduce the antibody into the substrate. The substrate after antibody introduction was washed with PBS. The substrate was fitted with a flat pipette tip manufactured by Fukami Chemical, and the change in fluorescence intensity on the substrate surface was measured using an automated dispensing device with an attached fluorescence microscope. The concentrations of the PC3 extracellular particle PBS solution were set to 0.03, 0.3, 3, and 30 fM. 1. Crystal mounting 2. Aspirate 150 μL of PBS and perform F0 measurement ×5 3. Aspirate 100 μL of sample (exosomes derived from PC3) 4. React for 5 min (25℃) 5. Complete ejection 6. Aspirate 150 μL of 10 mM PBS (140 mM NaCl, pH 7.4) 7. Automated fluorescence microscopy: Camera: Zyla 4.2; Filter: Cy5 (excitation wavelength 604-644 nm, fluorescence wavelength 672-712 nm); Objectives: ×5; Exposure time: 0.5 sec; Light intensity: 12%; Light source: mercury lamp 8. Repeat 3-7 times.

[0308] (Results) The results are shown in Figure 12. No response to extracellular bodies was generated in the substrate where pores were not formed without the use of silicon oxide particle nuclei.

[0309] (Example 14: Animal Experiment) This example demonstrates an empirical case based on animal experiments. Extracellular bodies in the blood of dogs or cats were measured. This experiment involved collecting blood from dogs or cats diagnosed by a veterinarian with cancer or internal organ disease and measuring extracellular bodies. After recovery, extracellular bodies were measured again, and the differences were observed to investigate whether cancer or internal organ disease in dogs or cats could be detected.

[0310] The following uses, in addition to those described above, are conceivable. Uses other than those for humans: 1. Examination of animals including pets (cats, dogs, birds, etc.) for cancer. 2. Detection of infectious pathogenic viruses or infectious proteins (prions, etc.) and other pathogenic / infectious agents carried by livestock (cattle, horses, pigs, chickens, etc.). 3. Detection of infectious pathogenic viruses or infectious proteins (prions, etc.) and other pathogenic / infectious agents carried by harmful animals (birds, bats, foxes, mongooses, raccoons, etc.). 4. Inspection of the quality or freshness of aquatic products. 5. Inspection of the quality or freshness of agricultural products. 6. Examination of pathogens and viruses in plants or trees, etc. (Example 15: Clinical Application) In this example, the following description provides evidence for its application in cancer diagnosis.

[0311] In clinical studies approved by the Clinical Research Review Committee of the hospitals conducting clinical trials, with patient consent, the presence or absence of cancer detection was investigated by observing the difference in sensor responses before and after total mastectomy to determine whether the sensor could be used to detect cancer. Further observation was conducted to investigate whether the sensor could detect cancer metastasis or recurrence. The therapeutic effect of drug therapy was investigated to determine whether the difference in sensor responses before and after drug administration could be used to evaluate it. Lifestyle-related diseases were investigated to determine whether a diagnosis could be made based on the relationship between food intake and sensor responses. Furthermore, a study was conducted to investigate whether companion diagnostics could be performed by inferring the presence of cancer cell surface antigens based on sensor responses. Finally, a study was conducted to investigate whether quality management could be implemented for cells used in cell therapy based on sensor responses.

[0312] In addition to the above, the technology of the present invention is envisioned to have the following applications: Cancer examination; Removal of residual cancer cells during cancer surgery; Examination of cancer metastasis or recurrence; Treatment efficacy of drug therapy; Examination of lifestyle-related diseases; Companion diagnostics for drug administration purposes; Cell quality management in cell engineering (Notes). As described above, the present invention has been illustrated using preferred embodiments, but it should be understood that the scope of the invention is to be interpreted only by the claims. In this specification, any patents, patent applications, and other documents cited are to be understood to be referenced in the same way as their contents are specifically described herein. [Industrial Applicability]

[0313] The technology provided in this invention can be applied to all fields (including diagnostics) that employ examination and analysis techniques. [Simplified Explanation of the Diagram]

[0010] Figure 1-1 shows the NMR spectrum of polymer E2. Figure 1-2 shows the NMR spectrum of polymer E3. Figure 1-3 shows the NMR spectrum of polymer E4. Figures 1-4a) to 1-4d) show the DLS measurement results of particles formed by integrating silicon oxide particles with each of polymers E2 to E4. Figures 1-5a) to 1-5d) show the Z-potential measurement results of particles formed by integrating silicon oxide particles with each of polymers E2 to E4. Figure 1-6 shows the DLS and Z-potential measurement results of particles formed by integrating silicon oxide particles with polymer (E2-0). Figure 2-1 shows the observation image of the particle-immobilized substrate obtained by fluorescence microscopy. Figure 2-2 shows the hybridization cell counting analysis results of the particle-immobilized substrate obtained by fluorescence microscopy. Figure 3-1 shows the observation image of the particle-immobilized substrate formed by integrating silicon oxide particles with each of polymers E4 obtained by fluorescence microscopy. Figure 3-2 shows an image obtained using fluorescence microscopy of the particle-immobilized substrate formed by integrating silicon oxide particles with various polymers E3. Figure 4 shows an image obtained using fluorescence microscopy of the particle-immobilized substrate (glass substrate). Figure 5-1 shows an image obtained using fluorescence microscopy of the particle-immobilized substrate (polystyrene particles). Figure 5-2 shows an image obtained using fluorescence microscopy of the particle-immobilized substrate (polystyrene particles). Figure 5-3 shows an image obtained using fluorescence microscopy of the particle-immobilized substrate (polystyrene particles). Figure 5-4 shows the hybridization cell counting analysis results obtained using fluorescence microscopy of the particle-immobilized substrate (polystyrene particles). Figure 6-1 shows an image obtained using fluorescence microscopy of the particle-immobilized substrate of Example 6. Figure 6-2 shows an image obtained using fluorescence microscopy after the polymer layer on the substrate of Example 6 has been polymerized. Figure 6-3 shows an image obtained using fluorescence microscopy after the silicon oxide nanoparticles on the substrate of Example 6 have been removed. Figure 6-4 shows the hybridization cell counting analysis results obtained by fluorescence microscopy on the particle-immobilized substrate. Figure 7 shows a fluorescence microscopy image of a substrate immobilized with a His-tagged polymer and silicon oxide nanoparticles. Figure 8 shows a fluorescence microscopy and SEM image of a substrate immobilized with a cationic silicon oxide nanoparticle and anionic polymer Ex8. Figure 9 shows a fluorescence microscopy and SEM image of a substrate immobilized with biodegradable nanoparticles. Figure 10-1 shows a fluorescence microscopy and SEM image of a substrate immobilized with a silicon oxide nanoparticle and a cationic polymer E2P20 containing aromatic components. Figure 10-2 shows a fluorescence microscopy image of a substrate immobilized with a polystyrene nanoparticle and a cationic polymer E2P20 containing aromatic components.Figure 11 shows fluorescence microscopy and SEM images of a substrate immobilized with His-tagged and thiol-group silicon oxide nanoparticles. Figure 12 shows the fluorescence intensity variation on the substrate surface of a sensor fabricated without the use of particles.

Claims

1. A substrate for fabricating an analytical sensor, comprising: A) a substrate body, and B) particles disposed in the substrate body in a single layer state.

2. The substrate for manufacturing an analytical sensor as claimed in claim 1, wherein the aforementioned particles are arranged without agglomeration.

3. The substrate for manufacturing an analytical sensor as claimed in claim 1 or 2, wherein the particles include particles integrated with the modifying substance.

4. The substrate for fabricating an analytical sensor as claimed in any one of claims 1 to 3, wherein the substrate further comprises C) a polymer matrix disposed on the substrate body, and the polymer matrix having at least a portion of a recess adapted to the object; wherein the particles are disposed in the recess.

5. A convex analysis sensor comprising: A) a substrate body; B) a polymer matrix disposed on the substrate body, wherein the polymer matrix has at least a portion of recesses adapted to a detection target, the particles being disposed in the recesses without agglomeration; C) a binding base for a signaling substance disposed on the particles; and D) a binding base for a specific binding molecule disposed on the particles to bind with a molecule that is a detection target; wherein the particles are disposed on the substrate body at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm².

6. An analytical sensor comprising: A) a substrate body; B) a polymer matrix disposed on the substrate body, wherein the polymer matrix has at least a portion of recesses adapted to a detection target; C) a binding base for a signal substance disposed on the recesses; and D) a binding base for a specific binding molecule disposed on the recesses to bind to a molecule that is a detection target; wherein the recesses are disposed on the substrate body at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm² without aggregation.

7. The analytical sensor as claimed in claim 6, wherein the aforementioned recesses exist without agglomeration at a density of 1×10⁴ particles / mm² to 1×10⁶ particles / mm².

8. A method for manufacturing a substrate for a convex analysis sensor, comprising: A) a step of providing particles; B) a step of adding the particles to the substrate in a manner that prevents the particles from agglomerating and disposing them on the substrate body; C) a step of providing the substrate with the particles fixed with a raw material for a polymer matrix; and D) a step of forming a substrate with the polymer matrix disposed thereon by supplying the substrate with conditions for polymerization of the polymer matrix.

9. A method for manufacturing a substrate for an analytical sensor, comprising: A) a step of providing particles; B) a step of adding the particles to the substrate in a manner that prevents the particles from agglomerating and disposing them on the substrate body; C) a step of providing the substrate to which the particles are fixed with a raw material for a polymer matrix; D) a step of forming a substrate disposed of the polymer matrix by providing the substrate with conditions for polymerization of the polymer matrix; and E) a step of forming recesses by providing conditions for the particles to dissociate from the substrate.

10. The method of claim 8 or 9, wherein the step of adding the particles in a non-agglomerated manner comprises adding the particles to the substrate at a concentration of 1.0 × 10⁰ particles / μL to 1.0 × 10¹⁰ particles / μL, or at an amount of 1.0 × 10⁰ particles / mm² to 1.0 × 10¹⁰ particles / mm² relative to the surface area of ​​the substrate.

11. A method for manufacturing a sensor for convex analysis, comprising: A) a step of providing particles; B) a step of adding the particles to a substrate in a manner that prevents the particles from agglomerating and disposing them on the substrate body; C) a step of providing a raw material for a polymer matrix to the substrate on which the particles are fixed; D) a step of forming a substrate with the polymer matrix disposed thereon by providing the substrate with conditions for polymerization of the polymer matrix; and E) a step of binding the analytical material to the particles.

12. A method for manufacturing an analytical sensor, comprising: A) a step of providing particles; B) a step of adding the particles to a substrate in a manner that prevents the particles from agglomerating and disposing them on the substrate body; C) a step of providing a raw material for a polymer matrix to the substrate on which the particles are fixed; D) a step of forming a substrate disposed of the polymer matrix by providing the substrate with conditions for polymerization of the polymer matrix; E) a step of forming recesses by providing conditions for the particles to dissociate from the substrate; and F) a step of binding an analytical substance to the recesses.

13. The method for manufacturing an analytical sensor as described in claim 12, wherein the analytical sensor is manufactured by forming recesses with a density of 1×10¹⁰ / mm² or less, and the coefficient of variation of the relative fluorescence intensity change in target material detection is 20% or less at a rate of 80% or more.

14. The method for manufacturing an analytical sensor as claimed in claim 12, wherein the analytical sensor is manufactured by forming recesses on a substrate with an area occupancy ratio of at least 80% of the recesses, and the coefficient of variation of the relative fluorescence intensity change in target material detection is at least 20%.

15. The method of any one of claims 8 to 14, wherein the step of non-agglomerated configuration comprises the step of configuring the particles on a substrate at a density of 1×10³ particles / mm² to 1×10¹⁰ particles / mm².

16. The method of any one of claims 8 to 15, wherein the aforementioned particles are in a single-layer state.

17. The method of any one of claims 9, 12 to 14, wherein in the step of forming the recess, the recess exists at a density of 1×10³ / mm² to 1×10¹⁰ / mm².

18. The method of any one of claims 8 to 17, wherein the step of configuring the particles comprises spin coating, dropping particles onto a substrate, immersing the substrate in a particle dispersion, lifting the substrate from the particle dispersion, or spraying the particle dispersion.

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