JPWO2019142913A1 - Fluorescent microparticles for glucose concentration mapping in 3D tissue - Google Patents

Abstract

【Task】
An object of the present invention is to provide a novel detection material and detection method capable of continuously measuring glucose inside a three-dimensional cell tissue such as spheroids in a simple, highly accurate and non-invasive manner.
SOLUTION:
Fluorescent microparticles for measuring glucose in three-dimensional cell tissue, core particles with an average particle size on the order of micrometers, a fluorescent dye unit that exhibits a fluorescent response by binding to glucose, and cell adhesion. Includes a cell adhesion unit having a molecule; the fluorescent microparticle having a structure in which the fluorescent dye unit and the cell adhesion unit are covalently immobilized on the surface of the core particle.
[Selection diagram] None

Description

The present invention relates to fluorescent microparticles capable of continuously measuring glucose existing in a three-dimensional cell tissue, and continuous glucose monitoring using the fluorescent microparticles.

Living tissues such as organs have a three-dimensional structure formed by cells. It is known that the biological functions of living tissues appear inside the tissues through cell-cell interactions and the surrounding environment. In recent years, 3D tissue construction technology has been developed in various fields such as regenerative medicine research aimed at replacing organs and tissues and "Organ on a chip" research for drug evaluation using human cell chips instead of animal experiments. It is attracting attention. In such three-dimensional tissue culture, cell aggregates such as spheroids are used, and monitoring of glucose concentration inside the spheroids and the like not only controls the environment of the culture system but also clarifies the mechanism of cell metabolism and the like. (For example, Non-Patent Document 1).

However, in the past, there have been few examples of observing the local environment inside three-dimensional cell tissues such as spheroids, and most of them have sharpened enzyme electrodes inserted into the cell aggregates, and glucose inside the cell aggregates is determined by the obtained current value. It is a measurement of the concentration. Such a method using an enzyme electrode has a limit in sharpening the electrode, and is easily affected by the surrounding environment such as pH and adsorption of proteins on the electrode surface, and has a problem in terms of accuracy. In addition to the invasion caused by the insertion of electrodes, there is concern about damage to cell aggregates because glucose is consumed during measurement and glucolactone and hydrogen peroxide are released.

C. C. Dibble and B. D. Manning, Nature Cell Biology 15, 555--564 (2013)

Therefore, it is an object of the present invention to provide a novel detection material and detection method capable of continuously measuring glucose inside a three-dimensional cell tissue such as spheroids in a simple, highly accurate and non-invasive manner. Is.

As a result of diligent studies to solve the above problems, the present inventors have made a fluorescent dye molecule and a target that show a fluorescent response by binding with glucose on the surface of fine particles having an average particle size on the order of micrometers. By covalently modifying a cell adhesion molecule capable of adhering to the three-dimensional cell tissue, glucose-responsive fluorescent microparticles that change the fluorescence intensity in response to an increase or decrease in glucose concentration can be produced. We have found that glucose inside three-dimensional cell tissues such as spheroids can be continuously monitored by using the fluorescent microparticles in a simple, highly accurate and non-invasive manner, and based on such findings, the present invention has been completed. It is a thing.

That is, the present invention, in one aspect,
<1> Fluorophore particles for measuring glucose in a three-dimensional cell tissue, core particles having an average particle size on the order of micrometers, a fluorescent dye unit that exhibits a fluorescent response by binding to glucose, and a fluorescent dye unit. Including a cell adhesion unit having cell adhesion molecules; the fluorescent dye unit and the cell adhesion unit have a structure covalently immobilized on the surface of the core particles.
The fluorescent microparticles;
<2> The fluorescent microparticles according to <1> above, wherein the core particles are selected from the group consisting of a silicon-containing material having a functional group for modification on the surface, a polymer polymer, and metal fine particles, respectively;
<3> The fluorescent microparticles according to <1> above, wherein the core particles are a porous material having a modifying functional group on the surface;
<4> One or more functional groups selected from the group consisting of a carboxyl group, a thiol group, an isocyano group, a thioisocyano group, an epoxy group, an active carboxylic acid ester, a maleimide group, an acetyl group, and an azide group. The fluorescent microparticle according to claim 2 or 3, which is a group;
<5> The fluorescent microparticle according to any one of <1> to <4>, wherein the core particle has an average particle size of 100 nm to 100 μm.
<6> The fluorescent microparticle according to any one of <1> to <5> above, wherein the cell adhesion unit contains a polypeptide or oligopeptide consisting of 4 or more amino acid residues;
<7> The above <1>, wherein the cell adhesion unit contains at least one selected from the group consisting of collagen, gelatin, proteoglycan, hyaluronic acid, fibronectin, laminin, tenascin, entactin, elastin, and compounds derived from them. > To the fluorescent microparticle according to any one of <5>;
<8> The fluorescent dye unit has a fluorescent group and a quenching group; in the absence of glucose, the fluorescent group is extinguished by the quenching group, but the fluorescent group is combined with glucose by the quenching group. The fluorescent microparticle according to any one of <1> to <7> above, which exhibits a fluorescence response by emitting light.
<9> The fluorescent microparticle according to <8> above, wherein the fluorescent group has anthracene and the quencher group has arylboronic acid;
<10> The above <1> to <9>, wherein the fluorescent dye unit has one or more amino groups at the terminal and is immobilized on the surface of the core particles by a covalent bond via the amino group. The fluorescent microparticle according to any one; and <11> the fluorescent dye unit is represented by the following formula (I) or (II).
(In formula (II), "PEG" represents a polyethylene glycol chain.)
The fluorescent microparticle according to any one of <1> to <10> is provided.

In another aspect, the invention
<12> A glucose monitoring sensor containing the fluorescent microparticles according to any one of <1> to <11>above;
<13> A glucose detection method comprising a step of detecting the presence of glucose in a three-dimensional cell tissue as a fluorescence response using the fluorescent microparticles according to any one of <1> to <11>above; and <14. > Provided is a method for continuously monitoring glucose, which comprises continuously monitoring the presence of glucose in a three-dimensional cell tissue by the method for detecting glucose according to the above <13>.

According to the present invention, the size of microparticles can be adjusted to a size suitable for a three-dimensional cell tissue such as spheroid to be measured, and the measurement can be performed without being affected by changes in the surrounding environment. It is possible to measure the concentration of glucose present with high accuracy. In addition, since glucose is not consumed during measurement and is measured in a non-contact manner, invasion in the three-dimensional cell tissue can be suppressed to a small extent, and it is possible to map the glucose concentration in the three-dimensional cell tissue in a more natural state. become.

In recent years, research on 3D tissue construction has become extremely prosperous for the purpose of regenerative medicine research aimed at replacing organs and tissues and drug evaluation using human cell chips instead of animal experiments. From the viewpoint, the significance of the present invention is great.

FIG. 1 is an electron microscope image and a fluorescence image image of the fluorescence microparticles of the present invention. These are (a) GF-particle, (b) GF-RGD-particle, and (c) GF-Col-particle. FIG. 2 is an SEM image of the fluorescent microparticles of the present invention. These are (a) GF-particle, (b) GF-RGD-particle, and (c) GF-Col-particle. FIG. 3 is a graph showing (a) a change in fluorescence spectrum when 0 to 1,000 mg / dL of glucose is added to the fluorescent microparticles of the present invention, and (b) a graph plotting a change in fluorescence intensity at 470 nm. .. FIG. 4 is a fluorescence image image when the fluorescent microparticles of the present invention are introduced into HepG2 (liver hepatocyte) spheroids. These are (a) GF-particle, (b) GF-RGD-particle, and (c) GF-Col-particle. FIG. 5 is an image obtained by monitoring the glucose concentration inside the spheroid using the fluorescent microparticles of the present invention.

Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these explanations, and other than the following examples, the scope of the present invention can be appropriately modified and implemented without impairing the gist of the present invention.

The fluorescent microparticles of the present invention are fluorescent microparticles for measuring glucose in a three-dimensional cell tissue.
1) Core particles with an average particle size on the order of micrometers and
2) A fluorescent dye unit that exhibits a fluorescent response by binding to glucose,
3) It is characterized by being composed of a cell adhesion unit having a cell adhesion molecule. Further, the fluorescent dye unit and the cell adhesion unit are characterized by having a structure immobilized by covalent bonds on the surface of the core particles. Generally, a "microparticle" is a particle having a particle size of submicron to submillimeter.

By having such a configuration, the fluorescent microparticles of the present invention have a size capable of invading the inside of a three-dimensional cell tissue such as a spheroid to be measured, and are stably adhered to the three-dimensional cell tissue by a cell adhesion unit. be able to. Since the surface of the core particle is provided with a fluorescent dye unit that changes the fluorescence intensity in response to an increase or decrease in glucose concentration, the inside of the three-dimensional cell tissue is not invaded by observing the fluorescence response. It is possible to measure and monitor the presence and concentration of glucose in a highly accurate and continuous manner. Here, the "three-dimensional cell tissue" is a three-dimensional (typically spherical) cell aggregate in which a plurality of cells are aggregated and aggregated, and is also called a cell aggregate or a spheroid.

Hereinafter, each component of the fluorescent microparticle of the present invention will be described.

1. 1. Core Particles Core particles are microparticles (microparticles) having an average particle size on the order of micrometers and are the base material for the fluorescent microparticles of the present invention. By appropriately changing the size of the core particles used, it is possible to adjust the fluorescent microparticles to a desired size. The average particle size of the core particles is 100 nm to 100 μm, preferably 1 to 20 μm, and more preferably 2 to 10 μm.

The material of the core particles is not particularly limited as long as it is a material that can be used for a living body such as a cell tissue, and is a material having a particle shape having a certain uniform particle size or processed into such a particle shape. It can be a possible material. Typically, as the core material, a silicon-containing material, a high molecular polymer, and metal fine particles can be used. For example, examples of the silicon-containing material include silica gel and zeolite. Further, examples of the high molecular polymer include polystyrene and nylon, and examples of the metal fine particles include gold (Au) fine particles. In addition, as the polymer polymer, polylactic acid, polyethylene, polyphenol, polyurethane, polyacrylic, benzoamine melamine, polycarbonate, polyolefin, polyester and the like can also be used.

From another point of view, the core particles are preferably a porous material. In the fluorescent microparticles of the present invention, the fluorescent dye unit is immobilized on the surface of the core particles, but by using a porous material, fluorescent molecules can also be immobilized inside the particles, whereby fluorescence It has the advantage that the binding between the molecule and glucose can be made less susceptible to the influence of the surrounding environment. Examples of such a porous material include an inorganic porous material such as mesoporous silica and zeolite, and a porous polymer material such as polystyrene beads. In addition, porous polymer materials such as polylactic acid, polyethylene, polyphenol, polyurethane, polyacrylic, benzoamine melamine, polycarbonate, polyolefin, and polyester can be used. When the core particles are a porous material, for example, the bulk density can be 3.0-4.5 g / cm ³ .

As described above, these core particles have a modifying functional group on the surface thereof for modifying and immobilizing the fluorescent dye unit and the cell adhesion unit. It is possible to form a covalent bond by reacting the modifying functional group on the surface of the core particle with the functional group existing at the end of the fluorescent dye unit or the like, and chemically fix these functional units to the surface of the core particle. it can. As an example of such a functional group for modification, one or more functional groups selected from the group consisting of a carboxyl group, a thiol group, an isocyano group, a thioisocyano group, an epoxy group, an active carboxylic acid ester, a maleimide group, an acetyl group, and an azide group. The group can be mentioned. For example, when the modifying functional group is a carboxyl group and the fluorescent dye unit or the cell adhesion unit has an amino group, these units are immobilized on the surface of the core particles by forming an amide bond. A method known in the art can be used to impart the modifying functional group onto the surface of the core particles.

Examples of such core particles having a functional group for modification on the surface include silica gel having a carboxyl group modified on the surface, polymer particles having a carboxyl group on the side chain, gold fine particles having a thiol group on the surface, and the like. .. However, it is not limited to these.

Since glucose to be measured has a hydroxyl group (OH group), it is preferable that the core particles do not have an OH group on the surface. For example, in the case of silica gel, the surface treatment can be performed by silane coupling.

2. Cell Adhesion Unit The cell adhesion unit imparts cell adhesion to the fluorescent microparticles of the present invention. As a result, when the fluorescent microparticles are close to the three-dimensional cell tissue, they can adhere to the cells and stably exist inside (or on the surface) of the cells, and the change in the fluorescence response due to the presence of glucose can be detected with higher accuracy. can do.

Preferably, the cell adhesion unit can contain a polypeptide or oligopeptide consisting of 4 or more amino acid residues as a cell adhesion factor. For example, the cell adhesion unit can include one or more selected from the group consisting of collagen, gelatin, proteoglycan, hyaluronic acid, fibronectin, laminin, tenascin, entactin, elastin, and compounds derived thereto. A mixture of these can also be used.

"Collagen" is one of the proteins that make up the dermis, ligaments, tendons, bones, cartilage, etc., and is an element that makes up the extracellular matrix. The peptide chain of collagen protein has a primary structure in which glycine repeats every 3 residues as "-(glycine)-(amino acid X)-(amino acid Y)-". In many types of collagen, three of these peptide chains gather to form a helical structure and are called tropocollagen. For example, one peptide chain of type I collagen has a sequence of repeating 1014 amino acid residues and has a molecular weight of about 100,000. It is known that there are more than 30 types of human collagen. For example, type I collagen is the main component in dermis, ligaments, tendons, bones, etc., and type II collagen is the main component in articular cartilage. In addition, the basement membrane, which is the backing structure of all epithelial tissues, mainly contains type IV collagen. Type I collagen is the most abundant in the body. Preferably, water-soluble collagen can be used. Further, "gelatin" means collagen extracted into water by heating with water for a long time. Gelatin may be peptided.

More preferably, the cell adhesion unit can use collagen, atelocollagen, gelatin, derivatives thereof and mixtures thereof derived from mammals or fish. "Atelocollagen" is collagen obtained by removing terror peptides existing at both ends of a collagen molecule by enzyme treatment, and is collagen that is soluble in water. Atelocollagen may be peptided.

The cell adhesion unit forms a covalent bond on the surface of the core particles by the reaction with the modifying functional group by the functional group originally contained therein or the functional group introduced by modification, and the surface of the core particle. Can be immobilized on top. Examples of the functional group of the cell adhesion unit include an amino group.

3. 3. Fluorescent dye unit The fluorescent microparticles of the present invention contain at least a fluorescent dye unit on the surface thereof that exhibits a fluorescent response by binding to glucose. Thereby, the presence of glucose in the three-dimensional cell tissue can be detected as a fluorescence response.

In the fluorescent dye unit, the fluorescent dye unit preferably has a fluorescent group and a quencher group. A "fluorescent group" is a site containing a molecule that fluoresces when excited at a specific wavelength. The "quenching group" is a site containing an electron acceptor that can reduce the emission from the fluorescent group by interacting with the fluorescent group, and the quenching action due to the binding with glucose is eliminated to form the fluorescent group. It causes light emission again. As a result, in the absence of glucose, the quencher group extinguishes the fluorescent group, but the fluorescent group emits light when the quencher group binds to glucose, so that the presence of glucose is turned on and off by a fluorescence response. Can be detected as.

Preferably, the fluorophore has anthracene and the quencher has arylboronic acid. However, as long as it exhibits a fluorescent response by binding to glucose, the present invention is not limited to these, and a combination of a fluorescent group and a quencher group known in the art can also be used.

Further, the fluorescent dye unit is fixed to the surface of the core particles by a covalent bond. Therefore, preferably, the fluorescent dye unit has a functional group capable of forming a covalent bond by a chemical reaction with the modifying functional group on the surface of the core particles. Typically, the fluorescent dye unit has one or more amino groups at its terminal, and is immobilized on the surface of fluorescent microparticles by a covalent bond between the amino group and the modifying functional group.

A preferable example of the fluorescent dye unit used in the fluorescent microparticles of the present invention is a compound represented by the following formula (I).

In the compound represented by the formula (I), the central atlasene is a fluorescent group, and the two arylboronic acids on both sides thereof are a quencher group. As shown in the equilibrium equation below, the fluorescence of atlasene is quenched by the arylboronic acid moiety in the absence of glucose, but when the arylboronic acid moiety and glucose are combined, the capacitance of the nitrogen atom and boron atom in the molecule is electrostatic. By the interaction, the electron transfer to the anthracene site is suppressed, the quenching action is eliminated, and the fluorescence emission of atlasene is generated. With such a recognition mechanism, the presence of glucose can be detected as an ON-OFF fluorescence response.

Further, the compound of the formula (I) has two amino groups at the terminals, and the amino groups fluoresce the fluorescent dye unit by reacting with the modifying functional group on the surface of the core particles. It can be immobilized on the surface of microparticles by covalent bond. Here, an example is shown in which the functional group at the terminal of the compound of the formula (I) is an amino group, but the present invention is not limited to this, and a functional group that can covalently bond with a modifying functional group on the surface of the core particle. It will be understandable to those skilled in the art that it can be used if.

Another preferred example of the fluorescent dye unit used in the fluorescent microparticles of the present invention is a compound represented by the following formula (II).

In formula (I), "PEG" represents a polyethylene glycol chain. The polyethylene glycol chain preferably has a repeating unit of 2 to 60. However, the length of the polyethylene glycol chain can be appropriately changed and used.

In the compound represented by the formula (II), similarly to the compound of the above formula (I), the atlasene in the central portion is a fluorescent group, and the two arylboronic acids on both sides thereof are a quenching group. Therefore, the mechanism for detecting the presence of glucose as an ON-OFF fluorescence response is as described above.

On the other hand, the compound represented by the formula (II) has two maleimide groups at the terminal unlike the compound of the above formula (I). By reacting the maleimide group with the modifying functional group on the surface of the core particles, the fluorescent dye unit can be covalently immobilized on the surface of the fluorescent microparticles.

4. Preparation of Fluorescent Microparticles The fluorescent microparticles of the present invention can be prepared by a gel preparation method known in the art. For example, since a microparticle material having a modifying functional group such as a carboxyl group on the surface is commercially available, as shown in Examples described later, such a microparticle material is used as a core particle, and a known amide condensing agent is used. The fluorescent microparticles of the present invention can be obtained by sequentially modifying and immobilizing the cell adhesion unit and the fluorescent dye unit on the surface of the core particles. The surface modification rates of the fluorescent dye unit and the cell adhesion unit can be appropriately adjusted. Examples of the amide condensing agent include DMT-MM.

5. Glucose detection and monitoring using fluorescent microparticles Further, the present invention uses the above-mentioned fluorescent microparticles to detect and monitor glucose in three-dimensional cell tissues such as spheroids, and a sensor containing the fluorescent microparticles. Also related to. Specifically, the fluorescent microparticles of the present invention are brought into contact with the three-dimensional cell tissue to be measured and irradiated with light of a specific wavelength to detect the presence of glucose in the three-dimensional cell tissue as a fluorescence response. be able to.

The fluorescent microparticles of the present invention can be introduced into a three-dimensional cell tissue by, for example, being contained in a culture medium in a three-dimensional cell culture to form a spheroid. Further, by producing a small sensor device including a light source such as an LED; a detector such as a photomultiplier tube; and a wireless data transfer system, and applying it to a culture system, it can be used as a continuous glucose monitoring sensor. For the detection of the fluorescence signal, a device or system known in the art such as a fluorescence measuring device or a microscope can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

1. 1. Preparation of Fluorescent Microparticles The fluorescent microparticles of the present invention were prepared using the following samples.
1) Core particles: polystyrene (average particle size 15 μm)
Cell adhesion unit: atelocollagen, RGD peptide amide condensing agent: DMT-MM (manufactured by the company)
Glucose-responsive fluorescent dye: Arylborone-oxidized anthracene derivative A (Product name "G55": manufactured by NARD Co., Ltd.)

2) Core particles: Nylon porous beads (average particle size 15 μm; bulk density 3.0 to 4.5 g / cm ³ )
Cell adhesion unit: atelocollagen, RGD peptide amide condensing agent: DMT-MM (manufactured by the company)
Glucose-responsive fluorescent dye: Arylborone-oxidized anthracene derivative A (Product name "G55": manufactured by NARD Co., Ltd.)

The following three types of fluorescent dye-immobilized microparticles were prepared using an amide condensing agent. Microparticles (GF-particle) in which only the glucose-responsive fluorescent dye is immobilized on the surface, microparticles (GF-RGD-particle) in which the fluorescent dye and RGD peptide are immobilized on the surface, fluorescent dye and atelocollagen are immobilized on the surface. Microparticles (GF-Col-particle). Both RGD peptide and atelocollagen are molecules having cell adhesion.

Specifically, each fluorescent microparticle was prepared by mixing microparticles and a collagen solution, adding DMT-MM as a condensing agent to the mixture, and then adding a fluorescent dye.

2. Evaluation of Glucose Response in Three-dimensional Cell Tissue by Fluorescent Microparticles The fluorescence response of glucose was evaluated using the three types of fluorescent microparticles obtained in Example 1.

Glucose-responsive dyes (GF dyes) contain anthracene, which acts as a specific glucose recognition site and as a fluorescence generation site, respectively. Therefore, as described above, the fluorescence is quenched in the absence of the glucose molecule, but when the glucose molecule binds to the diboronic acid moiety, it exhibits fluorescence emission.

First, an electron microscope image and a fluorescence image of fluorescent microparticles in a state before introduction into spheroids are shown in FIG. 1, and their SEM images are shown in FIG. All figures are (a) GF-particle, (b) GF-RGD-particle, and (c) GF-Col-particle. From these images, it can be seen that the fine particles have been modified with protein and homogeneous particles are leaning against them.

The change in the fluorescence spectrum when 0 to 1,000 mg / dL of glucose is added to these fluorescent microparticles is shown in FIG. 3 (a) (excitation wavelength: 405 nm). FIG. 3B is a plot of the change in fluorescence intensity at 470 nm. As a result, it was confirmed that the fluorescence intensity of the glucose-responsive dye increased as the glucose concentration increased.

Next, these fluorescent microparticles were introduced into the spheroids of HepG2 (liver hepatocytes) using an ultra-low adhesion multilayer plate, and a Lucor responsiveness test was performed. To monitor the glucose concentration, time-lapse video was used to observe the fluorescence intensity of the fluorescent microparticles.

FIG. 4 shows fluorescence images after introduction into spheroids. No binding to spheroids was observed in "GF-particle" containing no cell adhesion molecule. On the other hand, in "GF-RGD-particle" having RGD peptide, adhesion to spheroid was observed, but no increase in fluorescence intensity was observed. On the other hand, in "GF-Col-particle" having atelocollagen, an increase in fluorescence intensity was observed due to adhesion to spheroids and glucose. This suggests that the RGD peptide lacks stability as a cell adhesion molecule for glucose monitoring, and a peptide chain having a longer amino acid residue of 4 or more is effective.

Furthermore, the result of monitoring the glucose concentration inside the spheroid using "GF-Col-particle" is shown in FIG. 5 (after 0 to 2 hours have passed). As a result, the fluorescence intensity decreased with time, and glucose consumption could be observed over time. This demonstrates that the glucose concentration inside the spheroid can be suitably measured by using the "GF-Col-particle".

In the results shown in FIG. 5, the reduction rate of fluorescence was higher in the region closer to the center of the spheroid. It is probable that this was because the spheroid (D = 1 mm) was too large to move a sufficient amount of glucose to the center of the spheroid.

Claims (14)

Fluorescent microparticles for measuring glucose in three-dimensional cell tissue
Core particles with an average particle size on the order of micrometers and
A fluorescent dye unit that exhibits a fluorescent response by binding to glucose,
Includes cell adhesion units with cell adhesion molecules;
The fluorescent dye unit and the cell adhesion unit have a structure in which they are covalently immobilized on the surface of the core particles.
The fluorescent microparticle. The fluorescent microparticle according to claim 1, wherein the core particles are selected from the group consisting of a silicon-containing material having a functional group for modification on the surface, a polymer polymer, and metal fine particles. The fluorescent microparticle according to claim 1, wherein the core particle is a porous material having a modifying functional group on the surface. The modifying functional group is one or more functional groups selected from the group consisting of a carboxyl group, a thiol group, an isocyano group, a thioisocyano group, an epoxy group, an active carboxylic acid ester, a maleimide group, an acetyl group, and an azido group. , The fluorescent microparticle according to claim 2 or 3. The fluorescent microparticle according to any one of claims 1 to 4, wherein the core particles have an average particle size of 100 nm to 20 μm. The fluorescent microparticle according to any one of claims 1 to 5, wherein the cell adhesion unit contains a polypeptide or oligopeptide consisting of 4 or more amino acid residues. Claims 1 to 5, wherein the cell adhesion unit comprises one or more selected from the group consisting of collagen, gelatin, proteoglycan, hyaluronic acid, fibronectin, laminin, tenascin, entactin, elastin, and compounds derived thereto. The fluorescent microparticle according to any one. The fluorescent dye unit has a fluorescent group and a quenching group;
Any one of claims 1 to 7, wherein the quencher is extinguished by the quencher in the absence of glucose, but the fluorescent group emits light when the quencher binds to glucose. Fluorescent microparticles described in. The fluorescent microparticle according to claim 8, wherein the fluorescent group has anthracene and the quencher group has arylboronic acid. The one according to any one of claims 1 to 9, wherein the fluorescent dye unit has one or more amino groups at the terminal and is immobilized on the surface of the core particles by a covalent bond via the amino group. Fluorescent microparticles. The fluorescent dye unit is represented by the following formula (I) or (II).
(In formula (II), "PEG" represents a polyethylene glycol chain.)
The fluorescent microparticle according to any one of claims 1 to 10. A glucose monitoring sensor comprising the fluorescent microparticle according to any one of claims 1-11. A method for detecting glucose, which comprises a step of detecting the presence of glucose in a three-dimensional cell tissue as a fluorescent response using the fluorescent microparticles according to any one of claims 1 to 11. A method for continuously monitoring glucose, which comprises continuously monitoring the presence of glucose in a three-dimensional cell tissue by the method for detecting glucose according to claim 13.