WO2019142913A1 - Fluorescent microparticles for mapping glucose concentration inside three-dimensional tissue - Google Patents

Fluorescent microparticles for mapping glucose concentration inside three-dimensional tissue Download PDF

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WO2019142913A1
WO2019142913A1 PCT/JP2019/001475 JP2019001475W WO2019142913A1 WO 2019142913 A1 WO2019142913 A1 WO 2019142913A1 JP 2019001475 W JP2019001475 W JP 2019001475W WO 2019142913 A1 WO2019142913 A1 WO 2019142913A1
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glucose
fluorescent
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fluorescent microparticles
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昌治 竹内
淳 澤山
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国立大学法人東京大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the present invention relates to fluorescent microparticles capable of continuously measuring glucose present in three-dimensional cell tissue, and continuous glucose monitoring using the fluorescent microparticles.
  • Biological tissues such as organs have a three-dimensional structure formed by cells. Biological functions of living tissue are known to appear inside tissue through interactions between cells and the surrounding environment.
  • three-dimensional tissue construction techniques have 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. Has attracted attention.
  • cell aggregates such as spheroids are used, and monitoring of the glucose concentration inside the spheroids etc. not only controls the environment of the culture system, but also reveals the mechanism of cell metabolism etc. are also useful for the purpose (for example, Non-Patent Document 1).
  • an object of the present invention is to provide a novel detection material and detection method capable of continuously measuring glucose in a three-dimensional cell tissue such as a spheroid in a simple, highly accurate and non-invasive manner. It is.
  • the inventors of the present invention have found, on the surface of fine particles having an average particle size on the order of micrometers, a fluorescent dye molecule exhibiting a fluorescence response by binding to glucose, and a target By covalently modifying a cell adhesion molecule capable of adhering to three-dimensional cell tissue, it is possible to produce glucose-responsive fluorescent microparticles that change fluorescence intensity according to increase or decrease of glucose concentration, By using the said fluorescent microparticles, it discovers that glucose in the inside of three-dimensional cell tissue, such as a spheroid, can be continuously monitored simply, with high accuracy and non-invasiveness, and the present invention has been completed based on such findings. It is
  • the present invention ⁇ 1> A fluorescent microparticle for measuring glucose in a three-dimensional cell tissue, wherein the core particle has an average particle size of the order of micrometers, and a fluorochrome unit exhibiting a fluorescence response by binding with glucose, A cell adhesion unit having a cell adhesion molecule; the fluorochrome unit and the cell adhesion unit having a structure covalently immobilized on the surface of the core particle, Said fluorescent microparticles; ⁇ 2> The fluorescent microparticles according to ⁇ 1>, wherein the core particle is selected from the group consisting of a silicon-containing material, a polymer, and a metal fine particle each having a functional group for modification on the surface; ⁇ 3> The fluorescent microparticles according to ⁇ 1>, wherein the core particle is 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,
  • Fluorescent microparticles according to claim 2 or 3, which are groups. ⁇ 5> The fluorescent microparticles according to any one of ⁇ 1> to ⁇ 4>, wherein the core particle has an average particle diameter of 100 nm to 100 ⁇ m.
  • ⁇ 6> The fluorescent microparticles according to any one of the above ⁇ 1> to ⁇ 5>, wherein the cell adhesion unit comprises a polypeptide or an oligopeptide consisting of four or more amino acid residues; ⁇ 7> The ⁇ 1>, 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 therefrom.
  • the fluorescent microparticles according to any one of ⁇ 5>; ⁇ 8>
  • the fluorochrome unit has a fluorophore and a quencher; the fluorophore is quenched by the quencher in the absence of glucose, but the fluorophore is bound to glucose by the fluorophore.
  • the fluorescent microparticles according to any one of the above; and ⁇ 11> the fluorescent dye unit is represented by the following formula (I) or (II): (In the formula (II), “PEG” represents a polyethylene glycol chain.)
  • the fluorescent micro particle according to any one of the above ⁇ 1> to ⁇ 10> is provided.
  • the present invention ⁇ 12> A glucose monitoring sensor comprising the fluorescent microparticles according to any one of ⁇ 1> to ⁇ 11>above; ⁇ 13> A glucose detection method comprising the 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;> A continuous glucose monitoring method characterized by continuously monitoring the presence of glucose in a three-dimensional cell tissue by the glucose detection method described in ⁇ 13> above.
  • the size of the microparticles can be adjusted to a size suitable for three-dimensional cell tissue such as spheroid to be measured, and measurement can be performed without being affected by changes in the surrounding environment, The presence and concentration of glucose can be measured with high accuracy.
  • noncontact measurement is performed without consuming glucose at the time of measurement, it is possible to minimize the invasion in three-dimensional cell tissue, and to map the glucose concentration in three-dimensional cell tissue in a more natural state. become.
  • FIG. 1 is an electron microscope image and a fluorescence image of the fluorescent microparticles of the present invention.
  • A GF-particles,
  • B GF-RGD-particles,
  • c GF-Col-particles.
  • FIG. 2 is a SEM image of the fluorescent microparticles of the present invention.
  • A GF-particles,
  • B GF-RGD-particles,
  • c GF-Col-particles.
  • FIG. 3 is a graph showing (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 change in fluorescence intensity at 470 nm. .
  • FIG. 4 is a fluorescence image when the fluorescent microparticles of the present invention are introduced into HepG2 (hepatic liver cells) spheroids.
  • A GF-particles
  • B GF-RGD-particles
  • c GF-Col-particles.
  • FIG. 5 is an image obtained by monitoring the glucose concentration inside the spheroid using the fluorescent microparticles of the present invention.
  • the fluorescent microparticles of the present invention are fluorescent microparticles for measuring glucose in three-dimensional cell tissue, 1) Core particles having an average particle size on the order of micrometers, 2) A fluorochrome unit that exhibits a fluorescence response upon binding to glucose, 3) It is characterized by being comprised by the cell adhesion unit which has a cell adhesion molecule. Furthermore, the fluorescent dye unit and the cell adhesion unit are characterized in that they have a structure immobilized by covalent bonding on the surface of the core particle. In general, "microparticles" are particles having a particle size of submicron to submillimeter.
  • the fluorescent microparticles of the present invention have a size capable of entering into the interior of three-dimensional cell tissue such as spheroid to be measured, and stably adhere to the three-dimensional cell tissue by the cell adhesion unit. be able to. And, since the surface of the core particle is provided with a fluorescent dye unit that changes the fluorescence intensity according to the increase or decrease of the glucose concentration, the inside of the three-dimensional cell tissue without invading the three-dimensional cell tissue by observing the fluorescence response The presence and concentration of glucose can be measured and monitored continuously and accurately.
  • “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 spheroid.
  • Core particle is a microparticle (microparticulate) having an average particle size on the order of micrometers, and is a base of the fluorescent microparticle of the present invention. It is possible to adjust fluorescent microparticles to a desired size by appropriately changing the size of core particles used.
  • the average particle size of the core particles is 100 nm to 100 ⁇ m, preferably 1 to 20 ⁇ m, and more preferably 2 to 10 ⁇ m.
  • the material of the core particle is not particularly limited as long as it is a material that can be used for a living body such as cellular tissue, and it is processed into a material having a particle shape having a uniform particle diameter to some extent or such particle shape It can be a possible material.
  • silicon-containing materials, polymer polymers, and metal microparticles can be used as the core material.
  • silica gel, zeolite, etc. can be mentioned.
  • polystyrene, nylon, etc. can be mentioned as an example of a high molecular weight polymer
  • Gold (Au) microparticles can be mentioned as an example of metal particulates.
  • polylactic acid, polyethylene, polyphenol, polyurethane, polyacryl, benzoamine melamine, polycarbonate, polyolefin, polyester and the like can be used.
  • a core particle is a porous material from another viewpoint.
  • the fluorescent dye unit is immobilized on the surface of the core particle, but by using a porous material, fluorescent molecules can be immobilized also inside the particle, whereby fluorescence It offers the advantage of being less susceptible to the surrounding environment in binding molecules with glucose.
  • porous materials include inorganic porous materials such as mesoporous silica and zeolite, and porous polymer materials such as polystyrene beads.
  • porous polymer materials such as polylactic acid, polyethylene, polyphenol, polyurethane, polyacrylic, benzoamine melamine, polycarbonate, polyolefin and polyester can be used.
  • the core particle is a porous material, for example, the bulk density can be 3.0 to 4.5 g / cm 3 .
  • these core particles have a functional group for modification for modifying and immobilizing the fluorochrome unit and the cell adhesion unit on the surface thereof.
  • a functional group on the surface of the core particle is reacted with a functional group present at the end of the fluorescent dye unit or the like to form a covalent bond and chemically fix the functional unit to the surface of the core particle.
  • Such functional groups for modification include 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 activated carboxylic acid ester, a maleimide group, an acetyl group, and an azide group. Groups can be mentioned.
  • the functional group for modification is a carboxyl group and the fluorochrome unit and the cell adhesion unit have an amino group
  • these units are immobilized on the surface of the core particle by forming an amide bond.
  • Techniques known in the art can be used to provide the functional group for modification on the surface of the core particle.
  • core particles having a functional group for modification on the surface examples include silica gel with a carboxyl group modified on the surface, polymer particles with a carboxyl group in the side chain, and gold fine particles with a thiol group on the surface. . However, it is not limited to these.
  • glucose which is a measurement object is what has a hydroxyl group (OH group)
  • a core particle does not have OH group on the surface.
  • surface treatment can be performed by silane coupling.
  • the cell adhesion unit is to impart cell adhesion to the fluorescent microparticles of the present invention. Thereby, when the fluorescent microparticles are in proximity to a three-dimensional cell tissue, they can adhere to the cells and can be stably present inside (or on the surface), and the fluorescence response change due to the presence of glucose can be detected more accurately can do.
  • the cell adhesion unit can comprise, as a cell adhesion factor, a polypeptide or oligopeptide consisting of four or more amino acid residues.
  • 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 therefrom. Mixtures of these can also be used.
  • Collagen is one of the proteins constituting the dermis, ligaments, tendons, bones, cartilage and the like, and is an element constituting the extracellular matrix.
  • the peptide chain of collagen protein has a primary structure in which "-(glycine)-(amino acid X)-(amino acid Y)-" and glycine repeat every three residues. In many types of collagen, three peptide chains gather to form a helical structure and are called tropocollagen.
  • one peptide chain of type I collagen has a sequence repeating 1014 amino acid residues and has a molecular weight of about 100,000. It is known that there are 30 or more types of human collagen.
  • type I collagen is mainly used for dermis, ligaments, tendons, bones and the like
  • type II collagen is mainly used for articular cartilage
  • the basement membrane which is the lining structure of all epithelial tissues, mainly contains type IV collagen.
  • the most abundant in the body is type I collagen.
  • water soluble collagen can be used.
  • gelatin means what was extracted in water by heating with water for a long time among collagens. The gelatin may be peptided.
  • the cell adhesion unit can use mammalian or fish-derived collagen, atelocollagen, gelatin, their derivatives and mixtures thereof.
  • "Atelocollagen” is collagen obtained by enzymatic treatment of removing telopeptides present at both ends of a collagen molecule, and is collagen that has become soluble in water. Atelocollagen may be peptided.
  • the cell adhesion unit forms a covalent bond on the surface of the core particle by the reaction with the functional group for modification by the functional group inherently contained in them or the functional group introduced by modification, and the surface of the core particle is formed. It can be immobilized on top.
  • An amino group etc. are illustrated as a functional group of a cell adhesion unit.
  • the fluorescent microparticles of the present invention at least include a fluorescent dye unit that exhibits a fluorescent response upon binding to glucose. Thereby, the presence of glucose in the three-dimensional cell tissue can be detected as a fluorescence response.
  • the fluorochrome unit has a fluorophore and a quencher.
  • a "fluorophore” is a site that contains a molecule that fluoresces upon being excited at a particular wavelength.
  • the “quencher” is a site containing an electron acceptor capable of reducing the emission of light from the fluorophore by interaction with the fluorophore, and such quenching action is eliminated by binding to glucose, thereby making the fluorophore The light emission is caused again.
  • the fluorophore is quenched by the quencher in the absence of glucose, the presence of glucose is ON-OFF due to the fluorophore emitting light due to the binding of the quencher to glucose. It becomes possible to detect as
  • said fluorophore comprises anthracene and said quencher comprises arylboronic acid.
  • said quencher comprises arylboronic acid.
  • a fluorophore and a quencher known in the art can also be used.
  • the fluorescent dye unit is immobilized by covalent bonding on the surface of the core particle. 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 particle.
  • the fluorochrome unit has one or more amino groups at its end, and is immobilized on the surface of the fluorescent microparticles by covalent bonding of the amino group and the functional group for modification.
  • Preferred examples of the fluorescent dye unit used in the fluorescent microparticles of the present invention include compounds represented by the following formula (I).
  • the central atracene is a fluorophore and the two arylboronic acids on either side of it is a quencher.
  • the fluorescence of athracene is quenched by the arylboronic acid site in the absence of glucose, but when the arylboronic acid site is bound to glucose, the electrostatics of the nitrogen and boron atoms in the molecule are generated.
  • the interaction suppresses the electron transfer to the anthracene site, and the quenching action is eliminated, resulting in the emission of athracene fluorescence.
  • Such recognition mechanism allows the presence of glucose to be detected as an ON-OFF fluorescence response.
  • the compound of the formula (I) has two amino groups at the end, and the amino group reacts with the modifying functional group on the surface of the core particle to fluoresce the fluorescent dye unit. It can be immobilized on the surface of the microparticles by covalent bonding.
  • the terminal functional group of the compound of Formula (I) is an amino group was shown here, it is not limited to this, The functional group which can be covalently couple
  • Another preferable example of the fluorescent dye unit used in the fluorescent microparticles of the present invention includes a compound represented by the following formula (II).
  • PEG represents a polyethylene glycol chain.
  • the polyethylene glycol chain preferably has 2 to 60 repeating units. However, the length of the polyethylene glycol chain can be appropriately changed and used.
  • the compound represented by the formula (II) is different from the compound of the above formula (I) and has two maleimide groups at the terminal.
  • the fluorescent dye unit can be covalently immobilized on the surface of the fluorescent micro particle.
  • the fluorescent microparticles of the present invention can be prepared by gel production methods known in the art. For example, since a microparticulate material having a modifying functional group such as a carboxyl group on the surface is commercially available, such a microparticulate material is used as a core particle and a known amide condensing agent is used as shown in the examples described later.
  • the fluorescent microparticles of the present invention can be obtained by modifying and immobilizing the cell adhesion unit and the fluorescent dye unit sequentially on the surface of the core particle. The surface modification ratio of the fluorochrome unit and the cell adhesion unit can be appropriately adjusted.
  • an amide condensing agent DMT-MM can be mentioned.
  • the present invention further provides a method for detecting and monitoring glucose in three-dimensional cell tissue such as spheroids using the above-mentioned fluorescent microparticles, and a sensor including the fluorescent microparticles. It also relates to Specifically, the fluorescent microparticles of the present invention are brought into contact with the three-dimensional cell tissue to be measured, and the presence of glucose in the three-dimensional cell tissue is detected as a fluorescence response by irradiating light of a specific wavelength. be able to.
  • the fluorescent microparticles of the present invention can be introduced into three-dimensional cell tissue, for example, by forming them in a culture solution in three-dimensional cell culture to form spheroids.
  • a compact sensor device provided with a light source such as an LED, a detector such as a photomultiplier tube, a wireless data transfer system and the like can be manufactured and applied to a culture system to be used as a continuous glucose monitoring sensor.
  • the detection of a fluorescence signal can use apparatuses, systems, etc. well-known in the said technical field, such as a fluorescence measurement apparatus and a microscope.
  • microparticles immobilized on the surface of glucose responsive fluorochrome only
  • microparticles GF-RGD-particle
  • fluorochrome and RGD peptide are immobilized on the surface
  • fluorochrome and atelocollagen immobilized on the surface Microparticle
  • Both RGD peptide and atelocollagen are molecules having cell adhesion.
  • each fluorescent microparticle was prepared by the procedure of adding a fluorescent dye.
  • the glucose-responsive dye contains anthracene which acts as a specific glucose recognition site and as a fluorogenic site, respectively.
  • the fluorescence is quenched in the absence of the glucose molecule, but when the glucose molecule is attached to the diboronic acid moiety, it exhibits fluorescence.
  • FIG. 1 An electron microscope image and a fluorescence image of fluorescent microparticles in a state before introduction into a spheroid are shown in FIG. 1, and their SEM images are shown in FIG. All the figures are (a) GF-particles, (b) GF-RGD-particles, and (c) GF-Col-particles. From these images, it can be seen that the fine particles can be modified with protein and that homogeneous particles are supported.
  • FIG. 3 (a) (excitation wavelength: 405 nm). It is FIG. 3 (b) which plotted the fluorescence intensity change in 470 nm. As a result, it was confirmed that the glucose responsive dye increased in fluorescence intensity in response to an increase in glucose concentration.
  • these fluorescent microparticles were introduced into the HepG2 (hepatic liver cells) spheroids using an ultra-low adhesion multilayer plate, and a Lucose responsiveness test was performed. Time-lapse video was used to observe the fluorescence intensity of the fluorescent microparticles to monitor glucose concentration.
  • FIG. 5 shows the result of monitoring the glucose concentration inside the spheroid using “GF-Col-particle” (after 0 to 2 hours).
  • GF-Col-particles can be used to suitably measure the glucose concentration inside the spheroid.

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Abstract

[Problem] The present invention addresses the problem of providing a novel detection material and detection method which make it possible to conveniently, accurately and non-invasively perform continuous measurement of glucose inside three-dimensional cell tissues such as spheroids. [Solution] Fluorescent microparticles which measure glucose inside three-dimensional cell tissue, and include core particles, the average particle diameter of which is on the order of micrometers, a fluorescent pigment unit which exhibits a fluorescence response by bonding to glucose, and a cell adhesion unit having cell-adhesive molecules, wherein the fluorescent pigment unit and the cell adhesion unit are affixed by covalent bonds to the surface of the core particles.

Description

３次元組織内のグルコース濃度マッピングのための蛍光マイクロ粒子Fluorescent microparticles for glucose concentration mapping in three-dimensional tissues
　本発明は、３次元細胞組織内に存在するグルコースを連続的に測定可能な蛍光マイクロ粒子、及び当該蛍光マイクロ粒子を用いた連続的グルコースモニタリングに関する。 The present invention relates to fluorescent microparticles capable of continuously measuring glucose present in three-dimensional cell tissue, and continuous glucose monitoring using the fluorescent microparticles.
　臓器などの生体組織は、細胞により形成される３次元構造を有する。生体組織の生物学的機能は、細胞間の相互作用および周囲の環境を通して組織の内部に現れることが知られている。近年、臓器や組織の置換を目指した再生医療研究や動物実験に代わるヒト細胞のチップを用いた医薬品評価のための“Ｏｒｇａｎ ｏｎ ａ ｃｈｉｐ”研究など、様々な分野で３次元組織構築の技術が注目を集めている。かかる３次元組織培養においては、スフェロイドのような細胞凝集体が用いられ、当該スフェロイド等の内部におけるグルコース濃度のモニタリングは、培養系の環境を制御するだけでなく、細胞代謝のメカニズム等を明らかにするためにも有用である（例えば、非特許文献１）。 Biological tissues such as organs have a three-dimensional structure formed by cells. Biological functions of living tissue are known to appear inside tissue through interactions between cells and the surrounding environment. In recent years, three-dimensional tissue construction techniques have 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. Has attracted attention. In such three-dimensional tissue culture, cell aggregates such as spheroids are used, and monitoring of the glucose concentration inside the spheroids etc. not only controls the environment of the culture system, but also reveals the mechanism of cell metabolism etc. Are also useful for the purpose (for example, Non-Patent Document 1).
　しかしながら、従来、スフェロイド等の３次元細胞組織内部の局所的な環境を観察した例は少なく、そのほとんどが尖鋭化した酵素電極を細胞凝集体に挿し、得られる電流値により細胞凝集体内部のグルコース濃度を測定したものである。このような酵素電極を用いた方法は電極の尖鋭化に限界があり、ｐＨや電極表面へタンパク質の吸着など周囲の環境の影響を受けやすく精度点で問題があった。また、電極の挿入による侵襲のみならず、測定時にグルコースを消費し、グルコラクトンや過酸化水素を放出するため細胞凝集体へのダメージが懸念されていた。 However, conventionally, there have been few examples of observing the local environment inside a three-dimensional cell tissue such as spheroid, and most of the sharpened enzyme electrode is inserted into a cell aggregate, and the glucose value inside the cell aggregate is obtained according to the obtained current value. The concentration was measured. The method using such an enzyme electrode has a limit in sharpening of the electrode, and is susceptible to the influence of the surrounding environment such as pH and adsorption of a protein on the electrode surface, and there is a problem in accuracy. In addition to the invasion caused by the insertion of the electrode, there is concern that the cell aggregate may be damaged because it consumes glucose at the time of measurement and releases glucolactone and hydrogen peroxide.
　そこで、本発明は、簡便、高精度かつ非侵襲に、スフェロイド等の３次元細胞組織の内部におけるグルコースを連続的に計測可能な新規な検出用材料及び検出手法を提供することを課題とするものである。 Therefore, an object of the present invention is to provide a novel detection material and detection method capable of continuously measuring glucose in a three-dimensional cell tissue such as a spheroid in a simple, highly accurate and non-invasive manner. It is.
　本発明者らは、上記課題を解決するべく鋭意検討を行った結果、平均粒径がマイクロメートルのオーダーである微粒子の表面上に、グルコースとの結合により蛍光応答を示す蛍光色素分子、及び標的となる３次元細胞組織と接着することができる細胞接着性分子を共有結合により修飾することで、グルコース濃度の増減に対応し蛍光強度を変化させるグルコース応答性蛍光マイクロ粒子を作製することができ、当該蛍光マイクロ粒子を用いることで、簡便、高精度かつ非侵襲で、スフェロイド等の３次元細胞組織の内部におけるグルコースを連続的にモニタリングできることを見出し、かかる知見に基づき、本発明を完成するに至ったものである。 As a result of intensive studies to solve the above problems, the inventors of the present invention have found, on the surface of fine particles having an average particle size on the order of micrometers, a fluorescent dye molecule exhibiting a fluorescence response by binding to glucose, and a target By covalently modifying a cell adhesion molecule capable of adhering to three-dimensional cell tissue, it is possible to produce glucose-responsive fluorescent microparticles that change fluorescence intensity according to increase or decrease of glucose concentration, By using the said fluorescent microparticles, it discovers that glucose in the inside of three-dimensional cell tissue, such as a spheroid, can be continuously monitored simply, with high accuracy and non-invasiveness, and the present invention has been completed based on such findings. It is
　すなわち、本発明は、一態様において、
＜１＞３次元細胞組織内のグルコースを測定するための蛍光マイクロ粒子であって、平均粒径がマイクロメートルのオーダーであるコア粒子と、グルコースとの結合により蛍光応答を示す蛍光色素ユニットと、細胞接着性分子を有する細胞接着ユニットとを含み；前記蛍光色素ユニット及び前記細胞接着ユニットが、前記コア粒子の表面上に共有結合によって固定化された構造を有する、
該蛍光マイクロ粒子；
＜２＞前記コア粒子が、それぞれ表面上に修飾用官能基を有する含ケイ素材料、高分子ポリマー、及び金属微粒子よりなる群から選択される、上記＜１＞に記載の蛍光マイクロ粒子；
＜３＞前記コア粒子が、表面上に修飾用官能基を有する多孔質材料である、上記＜１＞に記載の蛍光マイクロ粒子；
＜４＞前記修飾用官能基が、カルボキシル基、チオール基、イソシアノ基、チオイソシアノ基、エポキシ基、活性カルボン酸エステル、マレイミド基、アセチル基、及びアジド基よりなる群から選択される１以上の官能基である、請求項２又は３に記載の蛍光マイクロ粒子；
＜５＞前記コア粒子が、１００ｎｍ～１００μｍの平均粒径を有する、上記＜１＞～＜４＞のいずれか１に記載の蛍光マイクロ粒子。
＜６＞前記細胞接着ユニットが、４以上のアミノ酸残基よりなるポリペプチド又はオリゴペプチドを含む、上記＜１＞～＜５＞のいずれか１に記載の蛍光マイクロ粒子；
＜７＞前記細胞接着ユニットが、コラーゲン、ゼラチン、プロテオグリカン、ヒアルロン酸、フィブロネクチン、ラミニン、テネイシン、エンタクチン、エラスチン、及びそれらに由来する化合物よりなる群から選択される１種以上を含む、上記＜１＞～＜５＞のいずれか１に記載の蛍光マイクロ粒子；
＜８＞前記蛍光色素ユニットが蛍光団及び消光団を有しており；グルコース非存在下では前記消光団により前記蛍光団が消光されているが、前記消光団がグルコースと結合することによって蛍光団が発光することにより蛍光応答を示す、上記＜１＞～＜７＞のいずれか１に記載の蛍光マイクロ粒子；
＜９＞前記蛍光団がアントラセンを有し、前記消光団がアリールボロン酸を有する、上記＜８＞に記載の蛍光マイクロ粒子；
＜１０＞前記蛍光色素ユニットが末端に１又は複数のアミノ基を有し、当該アミノ基を介した共有結合によって前記コア粒子の表面に固定化されている、上記＜１＞～＜９＞のいずれか１に記載の蛍光マイクロ粒子；及び
＜１１＞前記蛍光色素ユニットが以下の式（Ｉ）又は（ＩＩ）で表される、
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000004
 （式（ＩＩ）において、「ＰＥＧ」は、ポリエチレングリコール鎖を表す。）
上記＜１＞～＜１０＞のいずれか１に記載の蛍光マイクロ粒子を提供するものである。 That is, in one aspect, the present invention
<1> A fluorescent microparticle for measuring glucose in a three-dimensional cell tissue, wherein the core particle has an average particle size of the order of micrometers, and a fluorochrome unit exhibiting a fluorescence response by binding with glucose, A cell adhesion unit having a cell adhesion molecule; the fluorochrome unit and the cell adhesion unit having a structure covalently immobilized on the surface of the core particle,
Said fluorescent microparticles;
<2> The fluorescent microparticles according to <1>, wherein the core particle is selected from the group consisting of a silicon-containing material, a polymer, and a metal fine particle each having a functional group for modification on the surface;
<3> The fluorescent microparticles according to <1>, wherein the core particle is 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 activated carboxylic acid ester, a maleimide group, an acetyl group, and an azide group. 4. Fluorescent microparticles according to claim 2 or 3, which are groups.
<5> The fluorescent microparticles according to any one of <1> to <4>, wherein the core particle has an average particle diameter of 100 nm to 100 μm.
<6> The fluorescent microparticles according to any one of the above <1> to <5>, wherein the cell adhesion unit comprises a polypeptide or an oligopeptide consisting of four or more amino acid residues;
<7> The <1>, 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 therefrom. > The fluorescent microparticles according to any one of <5>;
<8> The fluorochrome unit has a fluorophore and a quencher; the fluorophore is quenched by the quencher in the absence of glucose, but the fluorophore is bound to glucose by the fluorophore. The fluorescent microparticles according to any one of the above <1> to <7>, which exhibit a fluorescence response by emitting light;
<9> The fluorescent microparticles according to <8>, wherein the fluorophore has anthracene, and the quencher has an arylboronic acid;
<10> The <1> to <9>, wherein the fluorescent dye unit has one or more amino groups at the end, and is immobilized on the surface of the core particle by covalent bonding via the amino group. The fluorescent microparticles according to any one of the above; and <11> the fluorescent dye unit is represented by the following formula (I) or (II):
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000004
 (In the formula (II), “PEG” represents a polyethylene glycol chain.)
The fluorescent micro particle according to any one of the above <1> to <10> is provided.
　別の態様において、本発明は、
＜１２＞上記＜１＞～＜１１＞のいずれか１に記載の蛍光マイクロ粒子を含む、グルコースモニタリングセンサー；
＜１３＞上記＜１＞～＜１１＞のいずれか１に記載の蛍光マイクロ粒子を用いて３次元細胞組織中のグルコースの存在を蛍光応答として検出する工程を含む、グルコース検出方法；及び
＜１４＞上記＜１３＞に記載のグルコース検出方法により、３次元細胞組織中のグルコースの存在を連続的にモニタリングすることを特徴とする、グルコースの連続的モニタリング方法を提供するものである。 In another aspect, the present invention
<12> A glucose monitoring sensor comprising the fluorescent microparticles according to any one of <1> to <11>above;
<13> A glucose detection method comprising the 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;> A continuous glucose monitoring method characterized by continuously monitoring the presence of glucose in a three-dimensional cell tissue by the glucose detection method described in <13> above.
　本発明によれば、測定対象であるスフェロイド等の３次元細胞組織内に適した大きさにマイクロ粒子のサイズを調整することができ、周囲の環境の変化に影響を受けずに測定できるため、高精度でグルコースの存在及ぶ濃度を測定することができる。また、測定時にグルコースを消費せずに非接触で測定するため、３次元細胞組織内における侵襲を小さく抑えられ、よりありのままに近い状態で、３次元細胞組織内におけるグルコース濃度をマッピングすることが可能になる。 According to the present invention, the size of the microparticles can be adjusted to a size suitable for three-dimensional cell tissue such as spheroid to be measured, and measurement can be performed without being affected by changes in the surrounding environment, The presence and concentration of glucose can be measured with high accuracy. In addition, since noncontact measurement is performed without consuming glucose at the time of measurement, it is possible to minimize the invasion in three-dimensional cell tissue, and to map the glucose concentration in three-dimensional cell tissue in a more natural state. become.
　臓器や組織の置換を目指した再生医療研究や動物実験に代わるヒト細胞のチップを用いた医薬品評価等を目的として３次元組織構築の研究が隆盛を極める昨今、３次元組織の評価系の構築という観点から本発明の意義は大きいものである。 Nowadays, three-dimensional tissue construction research is thriving with the aim of drug research using regenerative medicine research aimed at replacing organs and tissues, and human cell chips instead of animal experiments, etc. From the point of view, the significance of the present invention is great.
図１は、本発明の蛍光マイクロ粒子の電子顕微鏡イメージ及び蛍光イメージ画像である。（ａ）ＧＦ－ｐａｒｔｉｃｌｅ、（ｂ）ＧＦ－ＲＧＤ－ｐａｒｔｉｃｌｅ、（ｃ）ＧＦ－Ｃｏｌ－ｐａｒｔｉｃｌｅである。FIG. 1 is an electron microscope image and a fluorescence image of the fluorescent microparticles of the present invention. (A) GF-particles, (b) GF-RGD-particles, (c) GF-Col-particles. 図２は、本発明の蛍光マイクロ粒子のＳＥＭ画像である。（ａ）ＧＦ－ｐａｒｔｉｃｌｅ、（ｂ）ＧＦ－ＲＧＤ－ｐａｒｔｉｃｌｅ、（ｃ）ＧＦ－Ｃｏｌ－ｐａｒｔｉｃｌｅである。FIG. 2 is a SEM image of the fluorescent microparticles of the present invention. (A) GF-particles, (b) GF-RGD-particles, (c) GF-Col-particles. 図３は、本発明の蛍光マイクロ粒子に０～１，０００ｍｇ／ｄＬのグルコースを添加した場合の（ａ）蛍光スペクトル変化を示すグラフ、及び（ｂ）４７０ｎｍにおける蛍光強度変化をプロットしたグラフである。FIG. 3 is a graph showing (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 change in fluorescence intensity at 470 nm. . 図４は、ＨｅｐＧ２（肝臓肝細胞）スフェロイドに本発明の蛍光マイクロ粒子を導入した場合の蛍光イメージ画像である。（ａ）ＧＦ－ｐａｒｔｉｃｌｅ、（ｂ）ＧＦ－ＲＧＤ－ｐａｒｔｉｃｌｅ、（ｃ）ＧＦ－Ｃｏｌ－ｐａｒｔｉｃｌｅである。FIG. 4 is a fluorescence image when the fluorescent microparticles of the present invention are introduced into HepG2 (hepatic liver cells) spheroids. (A) GF-particles, (b) GF-RGD-particles, (c) GF-Col-particles. 図５は、本発明の蛍光マイクロ粒子を用いてスフェロイド内部におけるグルコース濃度のモニタリングを行い得られた画像である。FIG. 5 is an image obtained by monitoring the glucose concentration inside the spheroid using the fluorescent microparticles of the present invention.
　以下、本発明の実施形態について説明する。本発明の範囲はこれらの説明に拘束されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施することができる。 Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not restricted by these explanations, and it can be suitably changed and implemented in the range which does not spoil the meaning of the present invention also except the following examples.
　本発明の蛍光マイクロ粒子は、３次元細胞組織内のグルコースを測定するための蛍光マイクロ粒子であって、
１）平均粒径がマイクロメートルのオーダーであるコア粒子と、
２）グルコースとの結合により蛍光応答を示す蛍光色素ユニットと、
３）細胞接着性分子を有する細胞接着ユニット
により構成されることを特徴する。さらに、前記蛍光色素ユニット及び前記細胞接着ユニットが、前記コア粒子の表面上に共有結合によって固定化された構造を有することを特徴とするものである。一般に、「マイクロ粒子」とは、サブミクロンからサブミリメータの粒径を有する粒子のことである。 The fluorescent microparticles of the present invention are fluorescent microparticles for measuring glucose in three-dimensional cell tissue,
1) Core particles having an average particle size on the order of micrometers,
2) A fluorochrome unit that exhibits a fluorescence response upon binding to glucose,
3) It is characterized by being comprised by the cell adhesion unit which has a cell adhesion molecule. Furthermore, the fluorescent dye unit and the cell adhesion unit are characterized in that they have a structure immobilized by covalent bonding on the surface of the core particle. In general, "microparticles" are particles having a particle size of submicron to submillimeter.
　かかる構成を有することにより、本発明の蛍光マイクロ粒子は、測定対象となるスフェロイド等の３次元細胞組織の内部に侵入できるサイズを有し、細胞接着ユニットにより当該３次元細胞組織に安定に接着することができる。そして、コア粒子の表面にグルコース濃度の増減に対応して蛍光強度を変化させる蛍光色素ユニットを備えるため、その蛍光応答を観測することで３次元細胞組織を侵襲することなく、３次元細胞組織内部のグルコースの存在及び濃度を高精度かつ連続的に測定及びモニタリングすることができるというものである。ここで、「３次元細胞組織」とは、複数の細胞同士が集合・凝集化した３次元形状（典型的には球状）の細胞集合体であって、細胞凝集体或いはスフェロイドとも呼ばれる。 By having such a configuration, the fluorescent microparticles of the present invention have a size capable of entering into the interior of three-dimensional cell tissue such as spheroid to be measured, and stably adhere to the three-dimensional cell tissue by the cell adhesion unit. be able to. And, since the surface of the core particle is provided with a fluorescent dye unit that changes the fluorescence intensity according to the increase or decrease of the glucose concentration, the inside of the three-dimensional cell tissue without invading the three-dimensional cell tissue by observing the fluorescence response The presence and concentration of glucose can be measured and monitored continuously and accurately. Here, “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 spheroid.
　以下、本発明の蛍光マイクロ粒子の構成要素についてそれぞれ説明する。 The components of the fluorescent microparticles of the present invention will be described below.
１．コア粒子
　コア粒子は、マイクロメートルのオーダーの平均粒径を有するマイクロ粒子（マイクロ微粒子）であり、本発明の蛍光マイクロ粒子の基材となるものである。用いるコア粒子のサイズを適宜変更することで、蛍光マイクロ粒子を所望の大きさに調節することが可能である。コア粒子の平均粒径は、１００ｎｍ～１００μｍ、好ましくは１～２０μｍであり、より好ましくは、２～１０μｍである。 1. Core particle The core particle is a microparticle (microparticulate) having an average particle size on the order of micrometers, and is a base of the fluorescent microparticle of the present invention. It is possible to adjust fluorescent microparticles to a desired size by appropriately changing the size of core particles used. 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.
　コア粒子の材料は、細胞組織等の生体に対して用いることができる材料であれば特にその種類についての制限はなく、ある程度均一な粒径を有する粒子形状を有する材料、或いはかかる粒子形状に加工可能な材料であることができる。典型的には、コア材料としては、含ケイ素材料、高分子ポリマー、及び金属微粒子を用いることができる。例えば、含ケイ素材料としては、シリカゲルやゼオライトなどを挙げることができる。また、高分子ポリマーの例としては、ポリスチレンやナイロンなどを挙げることができ、金属微粒子の例としては、金（Ａｕ）微粒子を挙げることができる。その他にも、高分子ポリマーとして、ポリ乳酸、ポリエチレン、ポリフェノール、ポリウレタン、ポリアクリル、ベンゾアミンメラミン、ポリカーボネート、ポリオレフィン、ポリエステルなどを用いることもできる。 The material of the core particle is not particularly limited as long as it is a material that can be used for a living body such as cellular tissue, and it is processed into a material having a particle shape having a uniform particle diameter to some extent or such particle shape It can be a possible material. Typically, silicon-containing materials, polymer polymers, and metal microparticles can be used as the core material. For example, as a silicon-containing material, silica gel, zeolite, etc. can be mentioned. Moreover, polystyrene, nylon, etc. can be mentioned as an example of a high molecular weight polymer, Gold (Au) microparticles can be mentioned as an example of metal particulates. Besides, as the polymer, polylactic acid, polyethylene, polyphenol, polyurethane, polyacryl, benzoamine melamine, polycarbonate, polyolefin, polyester and the like can be used.
　また、別の観点から、コア粒子は、多孔質材料であることが好ましい。本発明の蛍光マイクロ粒子では、コア粒子の表面上に蛍光色素ユニットが固定化されるが、多孔質材料を用いることで、粒子内部にも蛍光分子を固定化することができ、これにより、蛍光分子とグルコースとの結合において周囲環境の影響を受け難くすることができるという利点を奏する。かかる多孔質材料としては、メソポーラスシリカやゼオライトなどの無機ポーラス材料、ポリスチレンビーズなどの多孔質ポリマー材料を挙げることができる。その他にも、ポリ乳酸、ポリエチレン、ポリフェノール、ポリウレタン、ポリアクリル、ベンゾアミンメラミン、ポリカーボネート、ポリオレフィン、ポリエステルなどの多孔質ポリマー材料を用いることができる。コア粒子が多孔質材料である場合、例えば、かさ密度は３．０～４．５ｇ／ｃｍ３であることができる。 Moreover, it is preferable that a core particle is a porous material from another viewpoint. In the fluorescent microparticles of the present invention, the fluorescent dye unit is immobilized on the surface of the core particle, but by using a porous material, fluorescent molecules can be immobilized also inside the particle, whereby fluorescence It offers the advantage of being less susceptible to the surrounding environment in binding molecules with glucose. Examples of such porous materials include inorganic porous materials such as mesoporous silica and zeolite, and porous polymer materials such as polystyrene beads. Besides, porous polymer materials such as polylactic acid, polyethylene, polyphenol, polyurethane, polyacrylic, benzoamine melamine, polycarbonate, polyolefin and polyester can be used. When the core particle is a porous material, for example, the bulk density can be 3.0 to 4.5 g / cm 3 .
　上述のように、これらコア粒子は、その表面上に蛍光色素ユニットと細胞接着ユニットを修飾して固定化するための修飾用官能基を有する。コア粒子表面上の修飾用官能基と、蛍光色素ユニット等の末端に存在する官能基が反応することで共有結合を形成させ、これら機能性ユニットを化学的にコア粒子の表面に固定することができる。かかる修飾用官能基の例としては、カルボキシル基、チオール基、イソシアノ基、チオイソシアノ基、エポキシ基、活性カルボン酸エステル、マレイミド基、アセチル基、及びアジド基よりなる群から選択される１以上の官能基を挙げることができる。例えば、修飾用官能基がカルボキシル基であり、蛍光色素ユニットや細胞接着ユニットがアミノ基を有する場合、これらユニットはアミド結合を形成することでコア粒子の表面上に固定化される。コア粒子の表面上への修飾用官能基の付与には、当該技術分野において公知の手法を用いることができる。 As described above, these core particles have a functional group for modification for modifying and immobilizing the fluorochrome unit and the cell adhesion unit on the surface thereof. A functional group on the surface of the core particle is reacted with a functional group present at the end of the fluorescent dye unit or the like to form a covalent bond and chemically fix the functional unit to the surface of the core particle. it can. Examples of such functional groups for modification include 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 activated carboxylic acid ester, a maleimide group, an acetyl group, and an azide group. Groups can be mentioned. For example, when the functional group for modification is a carboxyl group and the fluorochrome unit and the cell adhesion unit have an amino group, these units are immobilized on the surface of the core particle by forming an amide bond. Techniques known in the art can be used to provide the functional group for modification on the surface of the core particle.
　かかる表面上に修飾用官能基を有するコア粒子の例示としては、表面にカルボキシル基を修飾したシリカゲル、側鎖にカルボキシル基を有するポリマー粒子、表面にチオール基を有する金微粒子などを挙げることができる。ただし、これらに限定されるものではない。 Examples of core particles having a functional group for modification on the surface include silica gel with a carboxyl group modified on the surface, polymer particles with a carboxyl group in the side chain, and gold fine particles with a thiol group on the surface. . However, it is not limited to these.
　なお、測定対象であるグルコースがヒドロキシル基（ＯＨ基）を有するものであるため、コア粒子は表面上にＯＨ基を有しないことが好ましい。例えば、シリカゲルの場合には、シランカップリングによって表面処理を行うことができる。 In addition, since glucose which is a measurement object is what has a hydroxyl group (OH group), it is preferable that a core particle does not have OH group on the surface. For example, in the case of silica gel, surface treatment can be performed by silane coupling.
２．細胞接着ユニット
　細胞接着ユニットは、本発明の蛍光マイクロ粒子に細胞接着性を付与するものである。これにより、蛍光マイクロ粒子が３次元細胞組織に近接した場合に、当該細胞に接着しその内部（或いは表面）に安定に存在することができ、グルコースの存在による蛍光応答変化をより高精度に検出することができる。 2. Cell adhesion unit The cell adhesion unit is to impart cell adhesion to the fluorescent microparticles of the present invention. Thereby, when the fluorescent microparticles are in proximity to a three-dimensional cell tissue, they can adhere to the cells and can be stably present inside (or on the surface), and the fluorescence response change due to the presence of glucose can be detected more accurately can do.
　好ましくは、細胞接着ユニットは、細胞接着因子として、４以上のアミノ酸残基よりなるポリペプチド又はオリゴペプチドを含むことができる。例えば、細胞接着ユニットは、コラーゲン、ゼラチン、プロテオグリカン、ヒアルロン酸、フィブロネクチン、ラミニン、テネイシン、エンタクチン、エラスチン、及びそれらに由来する化合物よりなる群から選択される１種以上を含むことができる。これらの混合物を用いることもできる。 Preferably, the cell adhesion unit can comprise, as a cell adhesion factor, a polypeptide or oligopeptide consisting of four or more amino acid residues. 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 therefrom. Mixtures of these can also be used.
　「コラーゲン」とは、真皮、靭帯、腱、骨、軟骨などを構成するたんぱく質のひとつであり、細胞外マトリックスを構成する要素である。コラーゲンタンパク質のペプチド鎖は、“－（グリシン）－（アミノ酸Ｘ）－（アミノ酸Ｙ）－”と、グリシンが３残基ごとに繰り返す一次構造を有する。多くの型のコラーゲンでは、このペプチド鎖が３本集まり、らせん構造を形成し、トロポコラーゲンと呼ばれる。例えば、Ｉ型コラーゲンの１本のペプチド鎖は１０１４アミノ酸残基繰返す配列を持っており、分子量は１０万程度である。ヒトのコラーゲンには、３０種類以上あることが知られており、例えば、真皮、靱帯、腱、骨などではＩ型コラーゲンが、関節軟骨ではＩＩ型コラーゲンが主成分である。また、すべての上皮組織の裏打ち構造である基底膜にはＩＶ型コラーゲンが主に含まれている。体内で最も豊富に存在しているのはＩ型コラーゲンである。好ましくは、水可溶性コラーゲンを用いることができる。また、「ゼラチン」とは、コラーゲンのうち水で長時間加熱することで、水に抽出されたものを意味する。ゼラチンは、ペプチド化されていてもよい。 "Collagen" is one of the proteins constituting the dermis, ligaments, tendons, bones, cartilage and the like, and is an element constituting the extracellular matrix. The peptide chain of collagen protein has a primary structure in which "-(glycine)-(amino acid X)-(amino acid Y)-" and glycine repeat every three residues. In many types of collagen, three peptide chains gather to form a helical structure and are called tropocollagen. For example, one peptide chain of type I collagen has a sequence repeating 1014 amino acid residues and has a molecular weight of about 100,000. It is known that there are 30 or more types of human collagen. For example, type I collagen is mainly used for dermis, ligaments, tendons, bones and the like, and type II collagen is mainly used for articular cartilage. In addition, the basement membrane, which is the lining structure of all epithelial tissues, mainly contains type IV collagen. The most abundant in the body is type I collagen. Preferably, water soluble collagen can be used. Moreover, "gelatin" means what was extracted in water by heating with water for a long time among collagens. The gelatin may be peptided.
　より好ましくは、細胞接着ユニットは、哺乳動物または魚由来のコラーゲン、アテロコラーゲン、ゼラチン、それらの誘導体及びこれらの混合物を用いることができる。「アテロコラーゲン」とは、コラーゲン分子の両端に存在するテロペプチドを酵素処理で取り外したコラーゲンであり、水に可溶となったコラーゲンである。アテロコラーゲンは、ペプチド化されていてもよい。 More preferably, the cell adhesion unit can use mammalian or fish-derived collagen, atelocollagen, gelatin, their derivatives and mixtures thereof. "Atelocollagen" is collagen obtained by enzymatic treatment of removing telopeptides present at both ends of a collagen molecule, and is collagen that has become soluble in water. Atelocollagen may be peptided.
　細胞接着ユニットは、それらに本来的に含まれる官能基或いは修飾により導入された官能基によって、上記コア粒子の表面上に修飾用官能基との反応により共有結合を形成し、当該コア粒子の表面上に固定化されることができる。細胞接着ユニットの官能基としては、アミノ基等が例示される。 The cell adhesion unit forms a covalent bond on the surface of the core particle by the reaction with the functional group for modification by the functional group inherently contained in them or the functional group introduced by modification, and the surface of the core particle is formed. It can be immobilized on top. An amino group etc. are illustrated as a functional group of a cell adhesion unit.
３．蛍光色素ユニット
　本発明の蛍光マイクロ粒子は、グルコースとの結合により蛍光応答を示す蛍光色素ユニットを少なくともその表面に含む。これにより、３次元細胞組織内のグルコースの存在を蛍光応答として検出することができる。 3. Fluorescent Dye Unit The fluorescent microparticles of the present invention at least include a fluorescent dye unit that exhibits a fluorescent response upon binding to glucose. Thereby, the presence of glucose in the three-dimensional cell tissue can be detected as a fluorescence response.
　当該蛍光色素ユニットは、好ましくは、蛍光色素ユニットが蛍光団及び消光団を有している。「蛍光団」は、特定の波長で励起されることにより蛍光を発する分子を含む部位である。「消光団」は、当該蛍光団との相互作用により蛍光団からの発光を低減し得る電子受容体を含む部位であり、かつ、グルコースとの結合によりかかる消光作用が解消されて、蛍光団の発光を再び生じさせるものである。これにより、グルコース非存在下では前記消光団により前記蛍光団が消光されているが、前記消光団がグルコースと結合することによって蛍光団が発光することにより、グルコースの存在をＯＮ－ＯＦＦの蛍光応答として検出すること可能となる。 In the fluorochrome unit, preferably, the fluorochrome unit has a fluorophore and a quencher. A "fluorophore" is a site that contains a molecule that fluoresces upon being excited at a particular wavelength. The “quencher” is a site containing an electron acceptor capable of reducing the emission of light from the fluorophore by interaction with the fluorophore, and such quenching action is eliminated by binding to glucose, thereby making the fluorophore The light emission is caused again. Thus, although the fluorophore is quenched by the quencher in the absence of glucose, the presence of glucose is ON-OFF due to the fluorophore emitting light due to the binding of the quencher to glucose. It becomes possible to detect as
　好ましくは、前記蛍光団がアントラセンを有し、前記消光団がアリールボロン酸を有するものである。ただし、グルコースとの結合によって蛍光応答を示すものであれば、場合によっては、これらに限定されず、当該技術分野における公知の蛍光団と消光団の組み合わせを用いることもできる。 Preferably, said fluorophore comprises anthracene and said quencher comprises arylboronic acid. However, as long as it exhibits a fluorescence response upon binding to glucose, it is not limited in some cases, and a combination of a fluorophore and a quencher known in the art can also be used.
　また、当該蛍光色素ユニットは、前記コア粒子の表面に共有結合によって固定化されている。したがって、好ましくは、蛍光色素ユニットが、上記コア粒子の表面上の修飾用官能基と化学反応により共有結合を形成し得る官能基を有する。典型的には、蛍光色素ユニットは、その末端に１又は複数のアミノ基を有し、当該アミノ基と上記修飾用官能基との共有結合によって蛍光マイクロ粒子表面に固定化されている。 In addition, the fluorescent dye unit is immobilized by covalent bonding on the surface of the core particle. 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 particle. Typically, the fluorochrome unit has one or more amino groups at its end, and is immobilized on the surface of the fluorescent microparticles by covalent bonding of the amino group and the functional group for modification.
　本発明の蛍光マイクロ粒子において用いられる蛍光色素ユニットの好ましい例としては、以下の式（Ｉ）で表される化合物が挙げられる。
Figure JPOXMLDOC01-appb-C000005
 Preferred examples of the fluorescent dye unit used in the fluorescent microparticles of the present invention include compounds represented by the following formula (I).
Figure JPOXMLDOC01-appb-C000005
　式（Ｉ）で表される化合物において、中央部のアトラセンが蛍光団であり、その両側の２つのアリールボロン酸が消光団である。以下の平衡式に示すように、グルコース非存在下ではアリールボロン酸部位によりアトラセンの蛍光は消光されているが、アリールボロン酸部位とグルコースが結合すると、分子内の窒素原子とホウ素原子の静電相互作用によりアントラセン部位への電子移動が抑えられ、消光作用が解消してアトラセンの蛍光発光が生じる。かかる認識機構によって、グルコースの存在をＯＮ－ＯＦＦの蛍光応答として検出することができる。
Figure JPOXMLDOC01-appb-C000006
 In the compounds of formula (I), the central atracene is a fluorophore and the two arylboronic acids on either side of it is a quencher. As shown in the following equilibrium formula, the fluorescence of athracene is quenched by the arylboronic acid site in the absence of glucose, but when the arylboronic acid site is bound to glucose, the electrostatics of the nitrogen and boron atoms in the molecule are generated. The interaction suppresses the electron transfer to the anthracene site, and the quenching action is eliminated, resulting in the emission of athracene fluorescence. Such recognition mechanism allows the presence of glucose to be detected as an ON-OFF fluorescence response.
Figure JPOXMLDOC01-appb-C000006
　また、式（Ｉ）の化合物は、末端に２つのアミノ基を有しており、当該アミノ基が、上記コア粒子の表面上の修飾用官能基と反応することによって、当該蛍光色素ユニットを蛍光マイクロ粒子表面に共有結合により固定化することができる。なお、ここでは、式（Ｉ）の化合物の末端の官能基がアミノ基である例を示したが、これに限定されず、コア粒子の表面上の修飾用官能基と共有結合し得る官能基であれば用いることができることは当業者には理解可能であろう。 In addition, the compound of the formula (I) has two amino groups at the end, and the amino group reacts with the modifying functional group on the surface of the core particle to fluoresce the fluorescent dye unit. It can be immobilized on the surface of the microparticles by covalent bonding. In addition, although the example which the terminal functional group of the compound of Formula (I) is an amino group was shown here, it is not limited to this, The functional group which can be covalently couple | bonded with the functional group for modification on the surface of core particle Those skilled in the art will understand that it can be used.
　本発明の蛍光マイクロ粒子において用いられる蛍光色素ユニットの別の好ましい例としては、以下の式（ＩＩ）で表される化合物が挙げられる。
Figure JPOXMLDOC01-appb-C000007
 Another preferable example of the fluorescent dye unit used in the fluorescent microparticles of the present invention includes a compound represented by the following formula (II).
Figure JPOXMLDOC01-appb-C000007
　式（Ｉ）において、「ＰＥＧ」は、ポリエチレングリコール鎖を表す。当該ポリエチレングリコール鎖は、好ましくは、繰り返し単位が２～６０である。ただし、当該ポリエチレングリコール鎖の長さは、適宜変更して用いることができる。 In formula (I), "PEG" represents a polyethylene glycol chain. The polyethylene glycol chain preferably has 2 to 60 repeating units. However, the length of the polyethylene glycol chain can be appropriately changed and used.
　式（ＩＩ）で表される化合物も、上記式（Ｉ）の化合物と同様に、中央部のアトラセンが蛍光団であり、その両側の２つのアリールボロン酸が消光団である。したがって、グルコースの存在をＯＮ－ＯＦＦの蛍光応答として検出する機構は、上述のとおりである。 Also in the compound represented by the formula (II), similarly to the compound of the above formula (I), atracene in the central part is a fluorophore, and two arylboronic acids on both sides thereof are a quencher. Thus, the mechanism for detecting the presence of glucose as an ON-OFF fluorescence response is as described above.
　一方、式（ＩＩ）で表される化合物は、上記式（Ｉ）の化合物と異なり、末端に２つのマレイミド基を有している。当該マレイミド基が、上記コア粒子の表面上の修飾用官能基と反応することによって、当該蛍光色素ユニットを蛍光マイクロ粒子表面に共有結合により固定化することができる。 On the other hand, the compound represented by the formula (II) is different from the compound of the above formula (I) and has two maleimide groups at the terminal. By reacting the maleimide group with the modifying functional group on the surface of the core particle, the fluorescent dye unit can be covalently immobilized on the surface of the fluorescent micro particle.
４．蛍光マイクロ粒子の調製
　本発明の蛍光マイクロ粒子は、当該技術分野において公知のゲル作製方法により調製することができる。例えば、表面上にカルボキシル基等の修飾用官能基を有するマイクロ粒子材料は市販されているため、後述の実施例で示すように、かかるマイクロ粒子材料をコア粒子として用い、公知のアミド縮合剤を用いて、細胞接着ユニットと蛍光色素ユニットとを順にコア粒子の表面上に修飾・固定化することにより、本発明の蛍光マイクロ粒子を得ることができる。蛍光色素ユニットと細胞接着ユニットの表面修飾率は適宜調整することができる。アミド縮合剤の例としては、ＤＭＴ－ＭＭを挙げることができる。 4. Preparation of Fluorescent Microparticles The fluorescent microparticles of the present invention can be prepared by gel production methods known in the art. For example, since a microparticulate material having a modifying functional group such as a carboxyl group on the surface is commercially available, such a microparticulate material is used as a core particle and a known amide condensing agent is used as shown in the examples described later. The fluorescent microparticles of the present invention can be obtained by modifying and immobilizing the cell adhesion unit and the fluorescent dye unit sequentially on the surface of the core particle. The surface modification ratio of the fluorochrome unit and the cell adhesion unit can be appropriately adjusted. As an example of an amide condensing agent, DMT-MM can be mentioned.
５．蛍光マイクロ粒子を用いたグルコース検出及びモニタリング
　さらに、本発明は、上述の蛍光マイクロ粒子を用いて、スフェロイド等の３次元細胞組織内のグルコースを検出・モニタリングする方法、及び当該蛍光マイクロ粒子を含むセンサーにも関する。具体的には、本発明の蛍光マイクロ粒子を測定対象である３次元細胞組織と接触させ、特定の波長の光を照射することによって、３次元細胞組織内のグルコースの存在を蛍光応答として検出することができる。 5. Glucose Detection and Monitoring Using Fluorescent Microparticles The present invention further provides a method for detecting and monitoring glucose in three-dimensional cell tissue such as spheroids using the above-mentioned fluorescent microparticles, and a sensor including the fluorescent microparticles. It also relates to Specifically, the fluorescent microparticles of the present invention are brought into contact with the three-dimensional cell tissue to be measured, and the presence of glucose in the three-dimensional cell tissue is detected as a fluorescence response by irradiating light of a specific wavelength. be able to.
　本発明の蛍光マイクロ粒子は、例えば、３次元細胞培養における培養液中に含有させスフェロイドを形成することで、３次元細胞組織内に導入することができる。さらに、ＬＥＤ等の光源；光電子増倍管等の検出器；無線データ転送システム等を備える小型センサーデバイスを作製し、培養系に適用することで、連続的なグルコースモニタリングセンサーとして用いることができる。なお、蛍光シグナルの検出は、蛍光測定装置や顕微鏡など当該技術分野において公知の装置やシステム等を用いることができる。 The fluorescent microparticles of the present invention can be introduced into three-dimensional cell tissue, for example, by forming them in a culture solution in three-dimensional cell culture to form spheroids. Furthermore, a compact sensor device provided with a light source such as an LED, a detector such as a photomultiplier tube, a wireless data transfer system and the like can be manufactured and applied to a culture system to be used as a continuous glucose monitoring sensor. In addition, the detection of a fluorescence signal can use apparatuses, systems, etc. well-known in the said technical field, such as a fluorescence measurement apparatus and a microscope.
　以下、実施例により本発明をさらに詳細に説明するが、本発明はこれらによって限定されるものではない。 Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.
１．蛍光マイクロ粒子の調製
　以下の試料を用いて本発明の蛍光マイクロ粒子を調製した。
１）コア粒子：ポリスチレン（平均粒径１５μｍ）
　　細胞接着ユニット：アテロコラーゲン、ＲＧＤペプチド
　　アミド縮合剤：ＤＭＴ－ＭＭ（社製）
　　グルコース応答性蛍光色素：アリールボロン酸化アントラセン誘導体Ａ（製品名「G55」：株式会社ＮＡＲＤ社製）

２）コア粒子：ナイロン多孔質ビーズ（平均粒径15μｍ；かさ密度３．０～４．５ｇ／ｃｍ３）
　　細胞接着ユニット：アテロコラーゲン、ＲＧＤペプチド
　　アミド縮合剤：ＤＭＴ－ＭＭ（社製）
　　グルコース応答性蛍光色素：アリールボロン酸化アントラセン誘導体Ａ（製品名「G55」：株式会社ＮＡＲＤ社製）
Figure JPOXMLDOC01-appb-C000008
 1. Preparation of Fluorescent Microparticles The following samples were used to prepare fluorescent microparticles of the present invention.
1) Core particle: Polystyrene (average particle size 15 μm)
Cell adhesion unit: Atelocollagen, RGD peptide amide condensing agent: DMT-MM (manufactured by KK)
Glucose responsive fluorescent dye: arylboron oxidized anthracene derivative A (product name "G55": manufactured by NARD CORPORATION)

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 KK)
Glucose responsive fluorescent dye: arylboron oxidized anthracene derivative A (product name "G55": manufactured by NARD CORPORATION)
Figure JPOXMLDOC01-appb-C000008
　アミド縮合剤を用いて、以下の３種類の蛍光色素固定化マイクロ粒子を調製した。グルコース応答性蛍光色素のみ表面に固定化したマイクロ粒子（ＧＦ－ｐａｒｔｉｃｌｅ）、蛍光色素とＲＧＤペプチドを表面に固定化したマイクロ粒子（ＧＦ－ＲＧＤ－ｐａｒｔｉｃｌｅ）、蛍光色素とアテロコラーゲンを表面に固定化したマイクロ粒子（ＧＦ－Ｃｏｌ－ｐａｒｔｉｃｌｅ）。ＲＧＤペプチドおよびアテロコラーゲンは、いずれも細胞接着性を有する分子である。 The following three types of fluorescent dye-immobilized microparticles were prepared using an amide condensing agent. Microparticles (GF-particles) immobilized on the surface of glucose responsive fluorochrome only, microparticles (GF-RGD-particle) on which fluorochrome and RGD peptide are immobilized on the surface, fluorochrome and atelocollagen immobilized on the surface Microparticle (GF-Col-particle). Both RGD peptide and atelocollagen are molecules having cell adhesion.
　具体的には、マイクロ粒子とコラーゲン溶液を混ぜ、そこに縮合剤であるＤＭＴ－ＭＭを添加し、その後蛍光色素を添加する手順で各蛍光マイクロ粒子を調製した。 Specifically, the microparticles and the collagen solution were mixed, DMT-MM as a condensing agent was added thereto, and then each fluorescent microparticle was prepared by the procedure of adding a fluorescent dye.
２．蛍光マイクロ粒子による３次元細胞組織内のグルコース応答性の評価
　実施例１で得た３種の蛍光マイクロ粒子を用いてグルコースの蛍光応答を評価した。 2. Evaluation of Glucose Responsiveness in Three-Dimensional Cell Tissue by Fluorescent Microparticles The fluorescent fluorescence response of glucose was evaluated using the three types of fluorescent microparticles obtained in Example 1.
　グルコース応答性色素（ＧＦ色素）は、それぞれ特異的グルコース認識部位として作用する、及び蛍光発生部位として作用するアントラセンを含む。従って、上述のように、グルコース分子が存在しない場合には蛍光は消光されているが、グルコース分子がジボロン酸部分に結合すると、蛍光発光を示す。 The glucose-responsive dye (GF dye) contains anthracene which acts as a specific glucose recognition site and as a fluorogenic site, respectively. Thus, as described above, the fluorescence is quenched in the absence of the glucose molecule, but when the glucose molecule is attached to the diboronic acid moiety, it exhibits fluorescence.
　まず、スフェロイドに導入する以前の状態における蛍光マイクロ粒子の電子顕微鏡イメージ及び蛍光イメージを図１に、それらのＳＥＭ画像を図２に示す。いずれの図も、（ａ）ＧＦ－ｐａｒｔｉｃｌｅ、（ｂ）ＧＦ－ＲＧＤ－ｐａｒｔｉｃｌｅ、（ｃ）ＧＦ－Ｃｏｌ－ｐａｒｔｉｃｌｅである。これらの画像から、微粒子に蛋白質が修飾できており、均質な粒子がたもたれていることが分かる。 First, an electron microscope image and a fluorescence image of fluorescent microparticles in a state before introduction into a spheroid are shown in FIG. 1, and their SEM images are shown in FIG. All the figures are (a) GF-particles, (b) GF-RGD-particles, and (c) GF-Col-particles. From these images, it can be seen that the fine particles can be modified with protein and that homogeneous particles are supported.
　これら蛍光マイクロ粒子に０～１，０００ｍｇ／ｄＬのグルコースを添加した場合の蛍光スペクトル変化を図３（ａ）に示す（励起波長：４０５ｎｍ）。４７０ｎｍにおける蛍光強度変化をプロットしたものが図３（ｂ）である。この結果、グルコース応答性色素は、グルコース濃度の増加に応じて、蛍光強度が増大することを確認した。 The change in 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). It is FIG. 3 (b) which plotted the fluorescence intensity change in 470 nm. As a result, it was confirmed that the glucose responsive dye increased in fluorescence intensity in response to an increase in glucose concentration.
　次に、これら蛍光マイクロ粒子を、超低付着性多層プレートを用いてＨｅｐＧ２（肝臓肝細胞）のスフェロイド中に導入し、ルコース応答性試験を行った。グルコース濃度をモニターするために、タイムラプスビデオを用いて蛍光マイクロ粒子の蛍光強度を観察した。 Next, these fluorescent microparticles were introduced into the HepG2 (hepatic liver cells) spheroids using an ultra-low adhesion multilayer plate, and a Lucose responsiveness test was performed. Time-lapse video was used to observe the fluorescence intensity of the fluorescent microparticles to monitor glucose concentration.
　図４に、それぞれスフェロイドに導入後の蛍光イメージ画像を示す。細胞接着性分子を含まない「ＧＦ－ｐａｒｔｉｃｌｅ」では、スフェロイドとの結合は見られなかった。一方、ＲＧＤペプチドを有する「ＧＦ－ＲＧＤ－ｐａｒｔｉｃｌｅ」では、スフェロイドへの接着は見られたものの、蛍光強度の増大は見られなかった。これらに対し、アテロコラーゲンを有する「ＧＦ－Ｃｏｌ－ｐａｒｔｉｃｌｅ」では、スフェロイドに接着するとともに、グルコースとの結合による蛍光強度の増大が観測された。このことは、グルコースモニタリングのためには、細胞接着性分子としてＲＧＤペプチドでは安定性に欠き、より長い４以上のアミノ酸残基を有するペプチド鎖が有効であることを示唆するものである。 The fluorescence image image after introduce | transducing into a spheroid in FIG. 4 is shown, respectively. In the "GF-particles" containing no cell adhesion molecule, no binding to spheroids was observed. On the other hand, in the case of "GF-RGD-particles" having RGD peptide, adhesion to spheroids was observed, but no increase in fluorescence intensity was observed. On the other hand, in "GF-Col-particles" having atelocollagen, while adhering to the spheroid, an increase in fluorescence intensity due to binding to glucose was observed. This suggests that as a cell adhesion molecule, a peptide chain lacking the stability with RGD peptide and having longer four or more amino acid residues is effective for glucose monitoring.
　さらに、「ＧＦ－Ｃｏｌ－ｐａｒｔｉｃｌｅ」を用いて、スフェロイド内部におけるグルコース濃度のモニタリングを行った結果を図５に示す（０～２時間経過後）。その結果、蛍光強度は時間とともに減少し、グルコース消費を経時的に観測することができた。これは、「ＧＦ－Ｃｏｌ－ｐａｒｔｉｃｌｅ」を用いてスフェロイド内部のグルコース濃度を好適に測定できることを実証するものである。 Furthermore, FIG. 5 shows the result of monitoring the glucose concentration inside the spheroid using “GF-Col-particle” (after 0 to 2 hours). As a result, the fluorescence intensity decreased with time, and glucose consumption could be observed over time. This demonstrates that "GF-Col-particles" can be used to suitably measure the glucose concentration inside the spheroid.
　なお、図５に示す結果では、回転楕円体の中心に近い領域ほど蛍光の減少率が高かった。これは、スフェロイド（Ｄ＝１ｍｍ）が大きすぎて、十分な量のグルコースをスフェロイドの中心に移動させることができなかったことものと考えられる。 In the result shown in FIG. 5, the decrease rate of fluorescence was higher in the region closer to the center of the spheroid. It is believed that this is because the spheroid (D = 1 mm) was too large to transfer a sufficient amount of glucose to the center of the spheroid.

Claims (14)

  1. 　３次元細胞組織内のグルコースを測定するための蛍光マイクロ粒子であって、
    平均粒径がマイクロメートルのオーダーであるコア粒子と、
    グルコースとの結合により蛍光応答を示す蛍光色素ユニットと、
    細胞接着性分子を有する細胞接着ユニットとを含み；
    前記蛍光色素ユニット及び前記細胞接着ユニットが、前記コア粒子の表面上に共有結合によって固定化された構造を有する、
    該蛍光マイクロ粒子。     Fluorescent microparticles for measuring glucose in three-dimensional cell tissue,
    Core particles having an average particle size on the order of micrometers,
    A fluorochrome unit that exhibits a fluorescence response upon binding to glucose;
    A cell adhesion unit comprising a cell adhesion molecule;
    The fluorochrome unit and the cell adhesion unit have a structure covalently immobilized on the surface of the core particle,
    Said fluorescent microparticles.
  2. 　前記コア粒子が、それぞれ表面上に修飾用官能基を有する含ケイ素材料、高分子ポリマー、及び金属微粒子よりなる群から選択される、請求項１に記載の蛍光マイクロ粒子。 The fluorescent microparticles according to claim 1, wherein the core particle is selected from the group consisting of a silicon-containing material, a polymer, and a metal fine particle each having a modifying functional group on the surface.
  3. 　前記コア粒子が、表面上に修飾用官能基を有する多孔質材料である、請求項１に記載の蛍光マイクロ粒子。 The fluorescent microparticles according to claim 1, wherein the core particle is a porous material having a functional group for modification on the surface.
  4. 　前記修飾用官能基が、カルボキシル基、チオール基、イソシアノ基、チオイソシアノ基、エポキシ基、活性カルボン酸エステル、マレイミド基、アセチル基、及びアジド基よりなる群から選択される１以上の官能基である、請求項２又は３に記載の蛍光マイクロ粒子。 The modifying functional group is at least one functional group selected from the group consisting of carboxyl group, thiol group, isocyano group, thioisocyano group, epoxy group, activated carboxylic acid ester, maleimide group, acetyl group, and azide group The fluorescent microparticles according to claim 2 or 3.
  5. 　前記コア粒子が、１００ｎｍ～２０μｍの平均粒径を有する、請求項１～４のいずれか１に記載の蛍光マイクロ粒子。 The fluorescent microparticles according to any one of claims 1 to 4, wherein the core particles have an average particle size of 100 nm to 20 μm.
  6. 　前記細胞接着ユニットが、４以上のアミノ酸残基よりなるポリペプチド又はオリゴペプチドを含む、請求項１～５のいずれか１に記載の蛍光マイクロ粒子。 The fluorescent microparticles according to any one of claims 1 to 5, wherein the cell adhesion unit comprises a polypeptide or an oligopeptide consisting of 4 or more amino acid residues.
  7. 　前記細胞接着ユニットが、コラーゲン、ゼラチン、プロテオグリカン、ヒアルロン酸、フィブロネクチン、ラミニン、テネイシン、エンタクチン、エラスチン、及びそれらに由来する化合物よりなる群から選択される１種以上を含む、請求項１～５のいずれか１に記載の蛍光マイクロ粒子。 The cell adhesion unit according to any one of 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 therefrom. The fluorescent microparticles according to any one.
  8. 　前記蛍光色素ユニットが蛍光団及び消光団を有しており；
    グルコース非存在下では前記消光団により前記蛍光団が消光されているが、前記消光団がグルコースと結合することによって蛍光団が発光することにより蛍光応答を示す、請求項１～７のいずれか１に記載の蛍光マイクロ粒子。     The fluorochrome unit comprises a fluorophore and a quencher;
    The method according to any one of claims 1 to 7, wherein the fluorophore is quenched by the quencher in the absence of glucose, but the fluorescer emits a fluorescent response by binding to glucose. The fluorescent microparticles described in.
  9. 　前記蛍光団がアントラセンを有し、前記消光団がアリールボロン酸を有する、請求項８に記載の蛍光マイクロ粒子。 9. The fluorescent microparticle of claim 8, wherein the fluorophore comprises anthracene and the quencher comprises an aryl boronic acid.
  10. 　前記蛍光色素ユニットが末端に１又は複数のアミノ基を有し、当該アミノ基を介した共有結合によって前記コア粒子の表面に固定化されている、請求項１～９のいずれか１に記載の蛍光マイクロ粒子。 10. The fluorescent dye unit according to any one of claims 1 to 9, wherein the fluorescent dye unit has one or more amino groups at the end, and is immobilized on the surface of the core particle by covalent bonding via the amino group. Fluorescent microparticles.
  11. 　前記蛍光色素ユニットが以下の式（Ｉ）又は（ＩＩ）で表される、
    Figure JPOXMLDOC01-appb-C000001
    Figure JPOXMLDOC01-appb-C000002
     （式（ＩＩ）において、「ＰＥＧ」は、ポリエチレングリコール鎖を表す。）
    請求項１～１０のいずれか１に記載の蛍光マイクロ粒子。     The fluorescent dye unit is represented by the following formula (I) or (II):
    Figure JPOXMLDOC01-appb-C000001
    Figure JPOXMLDOC01-appb-C000002
     (In the formula (II), “PEG” represents a polyethylene glycol chain.)
    The fluorescent microparticles according to any one of claims 1 to 10.
  12. 　請求項１～１１のいずれか１に記載の蛍光マイクロ粒子を含む、グルコースモニタリングセンサー。 A glucose monitoring sensor comprising the fluorescent microparticles according to any one of claims 1-11.
  13. 　請求項１～１１のいずれか１に記載の蛍光マイクロ粒子を用いて３次元細胞組織中のグルコースの存在を蛍光応答として検出する工程を含む、グルコース検出方法。 A glucose detection method comprising the 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 claims 1 to 11.
  14. 　請求項１３に記載のグルコース検出方法により、３次元細胞組織中のグルコースの存在を連続的にモニタリングすることを特徴とする、グルコースの連続的モニタリング方法。 A continuous glucose monitoring method comprising continuously monitoring the presence of glucose in a three-dimensional cell tissue by the glucose detection method according to claim 13.
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