JP2011190403A - Fluorescent hydrogel fiber, method for producing the same, and saccharide-measuring sensor using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent hydrogel fiber excellent in detectability of saccharides such as glucose and having low invasiveness, and to provide a method for producing the same and a saccharide-measuring sensor using the same. <P>SOLUTION: The fluorescent hydrogel fiber contains a compound obtained by allowing 9,10-bis[[N-(6'-aminohexyl)-N-(2-boronobenzyl)amino]methyl]-2-acetylanthracene to react with acryloyl-(polyethylene glycol)-N-succinimide ester. There are also provided a method for producing the fluorescent hydrogel fiber and a saccharide-measuring sensor using the same. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a fluorescent hydrogel fiber, a method for producing the same, and a saccharide measurement sensor using the same.

  Implantable sensors are useful for monitoring the progress of disease states and monitoring therapeutic effects in various diseases, and are one of the fields that have been actively studied in recent years. In particular, in diabetes treatment, blood glucose control by continuous blood glucose measurement is said to contribute to the reduction of disease progression delay and morbidity of complications.

  Many current diabetic patients collect blood samples by puncturing their fingers or the like and supply them to a blood glucose meter to read the measured values for self-management of blood glucose. However, this method has problems in terms of pain and convenience for patients, and it is difficult to measure the blood glucose level frequently by measuring it several times a day. Currently. For these reasons, the usefulness of the implantable continuous blood glucose meter is considered high.

  On the other hand, technological development for continuously measuring the glucose concentration in the living body has been made for a long time. For example, the glucose concentration is measured by changing the amount of fluorescence using a substance that reversibly reacts with glucose and emits fluorescence. There is something to do. As such a fluorescent substance, Patent Document 1 has a fluorescent group, at least one phenylboronic acid moiety, and at least one amine nitrogen, and the amine nitrogen is in the vicinity of the phenylboronic acid moiety. Fluorescent compounds having a molecular structure that is disposed in the molecule and binds intramolecularly to the phenylboronic acid are disclosed. Patent Document 2 discloses an indicator component monomer having an excimer-forming polycyclic aromatic hydrocarbon such as a hydrophilic monomer and an anthracene borate derivative as an indicator polymer for detecting the concentration of an analyte in an aqueous environment. Copolymers are disclosed. Furthermore, Patent Document 3 discloses a method of directly immobilizing a fluorescent substance on a solid phase such as a plastic film as a fluorescent sensor.

  However, the sensor substances described in Patent Documents 1 to 3 have a film shape or a sheet shape, and there is a problem that there is not much invasion when the sensor substance is embedded in the body. For such a problem, Patent Document 4 proposes a saccharide measurement sensor in which a fluorescent sensor substance is fixed to a substrate such as a (meth) acrylamide film using a silane coupling agent or the like.

JP-A-8-53467 Japanese translation of PCT publication No. 2004-506069 US Pat. No. 6,319,540 JP 2006-104140 A

  However, the sugar measuring sensor described in Patent Document 4 requires room for improvement in terms of suppressing invasion as much as possible because it requires incision of the skin when it is implanted into the body and taken out.

  Accordingly, the present invention provides a fluorescent hydrogel fiber that is excellent in the ability to detect saccharides such as glucose and that is minimally invasive and can be easily implanted and removed from the body, a method for producing the same, and a saccharide measurement sensor using the same. For the purpose.

  As a result of intensive research, the present inventors have found that a fiber-shaped fluorescent hydrogel obtained by copolymerizing a fluorescent monomer compound and a polymerizable monomer containing a (meth) acrylamide residue can detect sugars such as glucose. And the present invention has been completed by finding that it can be implanted and removed from the body with less invasiveness.

  The fluorescent hydrogel fiber according to the present invention and a saccharide measurement sensor using the same are excellent in ability to detect saccharides in body fluids, and can be implanted into the body and taken out from the body in a minimally invasive manner.

FIG. 3 is a schematic diagram showing an aqueous solution column 12 filled in a tube 11. It is a schematic diagram which shows an example of the microchannel which produces an aqueous solution column. It is a schematic diagram which shows the cross section in the AA of FIG. 2A. It is a schematic diagram which shows the whole structure of a coaxial microfluidic device. It is a schematic diagram which expands and shows the principal part of FIG. 3A. It is the photograph of the bright field image which observed the fluorescence hydrogel fiber obtained in Example 3 with the fluorescence microscope. It is the photograph of the fluorescence image which observed the fluorescence hydrogel fiber obtained in Example 3 with the fluorescence microscope. It is a graph which shows the relationship between glucose concentration and the fluorescence intensity of fluorescent hydrogel fiber. It is the photograph of the bright field image which observed the fluorescence hydrogel fiber embedded in the ear | edge of a mouse | mouth with the fluorescence microscope. It is the photograph of the fluorescence image which observed the fluorescence hydrogel fiber embedded in the ear | edge of a mouse | mouth with the fluorescence microscope. It is the photograph of the fluorescence image which observed the ear of the mouse | mouth with the fluorescence microscope at the time of the blood glucose level of 114 mg / dL, the blood glucose level of 383 mg / dL, and the blood glucose level of 600 mg / dL or more. It is a figure showing the fluorescence intensity which analyzed the photograph of the fluorescence image of FIG. It is a schematic diagram which shows an example of the device for embedding fluorescent hydrogel fiber. It is a schematic diagram which shows an example of the method of embedding using the device for embedding fluorescent hydrogel fiber. In Example 5, it is the photograph of the bright-field image which observed the ear of the mouse | mouth which embedded the fluorescent hydrogel fiber using the device for embedding with the fluorescence microscope. In Example 5, it is the photograph of the fluorescence image which observed the ear of the mouse | mouth which embedded the fluorescence hydrogel fiber using the device for embedding with the fluorescence microscope. In Example 5, it is the photograph of the bright field image of the mouse | mouth ear after taking out the fluorescent hydrogel fiber embedded in the mouse | mouth ear. In Example 5, it is a photograph of the fluorescence image of the mouse ear after taking out the fluorescent hydrogel fiber embedded in the mouse ear. In Example 5, it is a photograph of the fluorescent hydrogel fiber taken out from the ear of the mouse.

  The first of the present invention is a fluorescent hydrogel fiber having a structure represented by the following chemical formula 1.

In Formula 1, X ₁ and X ₂ may be the same or different, and —COO—, —OCO—, —CH ₂ NR—, —NR—, —NRCO—, —CONR—, —SO ₂ NR— , —NRSO ₂ —, —O—, —S—, —SS—, —NRCOO—, —OCONR—, and —CO— containing at least one substituent selected from the group consisting of —CO— In which R is a hydrogen atom or a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms. .

  Specific examples of the linear or branched alkylene group having 1 to 30 carbon atoms include, for example, a methylene group, an ethylene group, a propylene group, an isopropylene group, a butylene group, an isobutylene group, a sec-butylene group, tert-butylene group, pentylene group, isopentylene group, neopentylene group, hexylene group, heptylene group, octylene group, nonylene group, decylene group, undecylene group, dodecylene group, tridecylene group, tetradecylene group, pentadecylene group, hexadecylene group, heptadecylene group Group, octadecylene group, nonadecylene group, eicosylene group, heicosylene group, docosylene group, tricosylene group, tetracosylene group, pentacosylene group, hexacosylene group, heptacosylene group, octacosylene group, nonacosylene group, or triacontylene group Etc., and the like. Preferably, it is a C3-C12 alkylene group, More preferably, they are a propylene group, a hexylene group, or an octylene group.

  The substituent contained in the alkylene group may be located at the end of the alkylene group or may be located inside the alkylene group. A preferred substituent is —NRCO— or —CONR—. R is a hydrogen atom or a linear or branched alkyl group having 1 to 10 carbon atoms, more preferably a hydrogen atom.

  Specific examples of the linear or branched alkyl group having 1 to 10 carbon atoms that can be used for R include, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, Isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, iso-amyl group, tert-pentyl group, neopentyl group, n-hexyl group, 3-methylpentan-2-yl group, 3-methyl Pentan-3-yl group, 4-methylpentyl group, 4-methylpentan-2-yl group, 1,3-dimethylbutyl group, 3,3-dimethylbutyl group, 3,3-dimethylbutan-2-yl group N-heptyl group, 1-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl group, 1-ethylpentyl group, 1- (n-propyl) butyl group, 1,1 Dimethylpentyl group, 1,4-dimethylpentyl group, 1,1-diethylpropyl group, 1,3,3-trimethylbutyl group, 1-ethyl-2,2-dimethylpropyl group, n-octyl group, 2-ethylhexyl Group, 2-methylhexan-2-yl group, 2,4-dimethylpentan-3-yl group, 1,1-dimethylpentan-1-yl group, 2,2-dimethylhexane-3-yl group, 2, 3-dimethylhexane-2-yl group, 2,5-dimethylhexane-2-yl group, 2,5-dimethylhexane-3-yl group, 3,4-dimethylhexane-3-yl group, 3,5- Dimethylhexane-3-yl group, 1-methylheptyl group, 2-methylheptyl group, 5-methylheptyl group, 2-methylheptan-2-yl group, 3-methylheptan-3-yl group, 4-methylheptyl group Tan-3-yl group, 4-methylheptan-4-yl group, 1-ethylhexyl group, 2-ethylhexyl group, 1-propylpentyl group, 2-propylpentyl group, 1,1-dimethylhexyl group, 1,4 -Dimethylhexyl group, 1,5-dimethylhexyl group, 1-ethyl-1-methylpentyl group, 1-ethyl-4-methylpentyl group, 1,1,4-trimethylpentyl group, 2,4,4-trimethyl Pentyl group, 1-isopropyl-1,2-dimethylpropyl group, 1,1,3,3-tetramethylbutyl group, n-nonyl group, 1-methyloctyl group, 6-methyloctyl group, 1-ethylheptyl group 1- (n-butyl) pentyl group, 4-methyl-1- (n-propyl) pentyl group, 1,5,5-trimethylhexyl group, 1,1,5-trimethylhexyl group , 2-methyloctane-3-yl group, n-decyl group, 1-methylnonyl group, 1-ethyloctyl group, 1- (n-butyl) hexyl group, 1,1-dimethyloctyl group, or 3,7- A dimethyloctyl group etc. are mentioned. More preferably, it is a linear or branched alkyl group having 1 to 5 carbon atoms.

In Formula 1, Z ₁ and Z ₂ may be the same or different and are —O— or —NR′—, wherein R ′ is a hydrogen atom or a substituted or unsubstituted carbon number of 1 -10 alkyl groups. Z ₁ and Z ₂ are more preferably —O—.

  Specific examples of the linear or branched alkyl group having 1 to 10 carbon atoms that can be used for R ′ are the same as described above, and thus the description thereof is omitted here. More preferably, it is a linear or branched alkyl group having 1 to 5 carbon atoms.

  Q, Q ′, Q ″ and Q ′ ″ may be the same or different, and are a hydrogen atom, a hydroxy group, a substituted or unsubstituted C 1-10 linear or branched An alkyl group, an acyl group having 2 to 11 carbon atoms, a substituted or unsubstituted linear or branched alkoxy group having 1 to 10 carbon atoms, a group containing a halogen atom, a carboxyl group, a carboxylic acid ester group , A carboxylic acid amide group, a cyano group, a nitro group, an amino group, or an alkylamino group having 1 to 10 carbon atoms.

  Specific examples of the linear or branched alkyl group having 1 to 10 carbon atoms are the same as described above, and thus the description thereof is omitted here.

  The acyl group having 2 to 11 carbon atoms is preferably a group represented by the following chemical formula 2.

  In Chemical Formula 2, L is a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms.

  Examples of the acyl group having 2 to 11 carbon atoms include an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a valeryl group, and an isovaleryl group. The number of carbon atoms of the L alkyl group in the chemical formula 2 is preferably 1 to 4, more preferably 1 (that is, an acetyl group). By introducing an acyl group into the anthracene residue, an effect of increasing the interval between the excitation wavelength and the maximum fluorescence wavelength can be obtained.

  Examples of the linear or branched alkoxy group having 1 to 10 carbon atoms include, for example, a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, and a sec-butoxy group. Tert-butoxy group, n-pentyloxy group, isopentyloxy group, neopentyloxy group, 1,2-dimethyl-propoxy group, n-hexyloxy group, 3-methylpentan-2-yloxy group, 3-methyl Pentane-3-yloxy group, 4-methylpentyloxy group, 4-methylpentan-2-yloxy group, 1,3-dimethylbutyloxy group, 3,3-dimethylbutyloxy group, 3,3-dimethylbutane-2 -Yloxy group, n-heptyloxy group, 1-methylhexyloxy group, 3-methylhexyloxy group, 4-methylhexyl Oxy group, 5-methylhexyloxy group, 1-ethylpentyloxy group, 1- (n-propyl) butyloxy group, 1,1-dimethylpentyloxy group, 1,4-dimethylpentyloxy group, 1,1-diethyl Propyloxy group, 1,3,3-trimethylbutyloxy group, 1-ethyl-2,2-dimethylpropyloxy group, n-octyloxy group, 2-ethylhexyloxy group, 2-methylhexane-2-yloxy group, 2,4-dimethylpentan-3-yloxy group, 1,1-dimethylpentan-1-yloxy group, 2,2-dimethylhexane-3-yloxy group, 2,3-dimethylhexane-2-yloxy group, 2, 5-dimethylhexane-2-yloxyoxy group, 2,5-dimethylhexane-3-yloxy group, 3,4-dimethyl San-3-yloxy group, 3,5-dimethylhexane-3-yloxy group, 1-methylheptyloxy group, 2-methylheptyloxy group, 5-methylheptyloxy group, 2-methylheptan-2-yloxy group, 3-methylheptane-3-yloxy group, 4-methylheptane-3-yloxy group, 4-methylheptan-4-yloxy group, 1-ethylhexyloxy group, 2-ethylhexyloxy group, 1-propylpentyloxy group, 2 -Propylpentyloxy group, 1,1-dimethylhexyloxy group, 1,4-dimethylhexyloxy group, 1,5-dimethylhexyloxy group, 1-ethyl-1-methylpentyloxy group, 1-ethyl-4- Methylpentyloxy group, 1,1,4-trimethylpentyloxy group, 2,4,4-trimethylpe Nyloxy group, 1-isopropyl-1,2-dimethylpropyloxy group, 1,1,3,3-tetramethylbutyloxy group, n-nonyloxy group, 1-methyloctyloxy group, 6-methyloctyloxy group, 1 -Ethyl heptyloxy group, 1- (n-butyl) pentyloxy group, 4-methyl-1- (n-propyl) pentyloxy group, 1,5,5-trimethylhexyloxy group, 1,1,5-trimethyl Hexyloxy group, 2-methyloctane-3-yloxy group, n-decyloxy group, 1-methylnonyloxy group, 1-ethyloctyloxy group, 1- (n-butyl) hexyloxy group, 1,1-dimethyloctyl Examples thereof include an oxy group and a 3,7-dimethyloctyloxy group.

  Examples of the group containing a halogen atom include, for example, F-, Cl-, Br-, I-, OI- (iodooxy group), or a linear or branched group having 1 to 10 carbon atoms substituted with halogen. And the like.

  Examples of the alkylamino group having 1 to 10 carbon atoms include, for example, methylamino group, ethylamino group, n-propylamino group, isopropylamino group, n-butylamino group, n-pentylamino group, and n-hexylamino. Group, n-octylamino group, n-decylamino group, n-isoamylamino group and the like.

  Further, at least one of Q and Q 'and Q "and Q"' may be bonded to each other to form an aromatic ring or a heterocyclic ring. Examples of the aromatic ring include a benzene ring. Examples of the heterocyclic ring include, for example, pyrazole ring, pyrrole ring, furan ring, thiophene ring, imidazole ring, thiazole ring, isothiazole ring, oxazole ring, isoxazole ring, pyridine ring, pyrazine ring, pyrimidine ring, Examples include pyridazine ring.

  When at least one of Q, Q ′, Q ″, and Q ′ ″ introduces a nitro group, a cyano group, or an acyl group, it contributes to the red transition of fluorescence or the increase in the interval between the excitation wavelength peak and the fluorescence wavelength peak. May be preferable.

  In the present invention, at least one of Q, Q ′, Q ″, and Q ′ ″ is a nitro group, a cyano group, or an acyl group having 2 to 11 carbon atoms represented by Formula 2 above. It is preferably a nitro group, a cyano group or an acetyl group, more preferably an acetyl group. In addition, it is preferable that 1-3 of Q, Q ′, Q ″, and Q ′ ″ are substituted with the above substituents, more preferably 1 to 2, and even more preferably 1 It is a piece.

Y ₁ and Y ₂ may be the same or different and may be a divalent organic residue which may be substituted. Y ₁ and Y ₂ preferably have such hydrophilicity that the fluorescent monomer compound can be rendered water-soluble. Here, hydrophilicity to the extent that the fluorescent monomer compound can be made water-soluble means that it dissolves in water in the concentration range necessary for polymerizing the fluorescent monomer compound without the presence of an organic solvent or solubilizer. To do. Specific examples of the group used for Y ₁ and Y ₂ include, for example, an amino group, a carbonyloxy group, or a hydrophilic group such as a sulfonic acid group, a nitro group, an amino group, a phosphoric acid group, or a hydroxy group. And a divalent organic residue having a hydrophilic bond such as an ether bond, an amide bond, or an ester bond in the structure.

At least one of Y ₁ and Y ₂ preferably contains a group represented by the following chemical formula 3 or the following chemical formula 4. Further, at least one of Y ₁ and Y ₂ may contain both a group represented by the following chemical formula 3 and a group represented by the following chemical formula 4, and in this case, a group represented by the following chemical formula 3 and The arrangement of the group represented by the following chemical formula 4 may be a block shape or a random shape. Furthermore, you may have another said substituent and a bivalent organic residue.

  In the chemical formula 3 and the chemical formula 4, n is 2 to 5, preferably 2 to 4, more preferably 2 or 3. Moreover, j is 1-5, Preferably it is 1-3, More preferably, it is 1. Furthermore, m is 1 to 200, preferably 20 to 150, and more preferably 40 to 120. In addition, * in the said Chemical formula 3 and the said Chemical formula 4 represents a bonding point.

The molecular weight of the Y ₁ and Y ₂ moieties is preferably 500 to 10,000, and more preferably 1,000 to 5,000. The divalent organic residue represented by the chemical formula 2 or the chemical formula 3 can be formed, for example, by polymerizing alkylene glycol such as ethylene glycol or propylene glycol, or vinyl alcohol.

A ₁ and A ₂ may be the same or different and are a hydrogen atom or a methyl group.

U ₁ , U ₂ , U ₃ and U ₄ may be the same or different and are a hydrogen atom or a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms. .

  In the present specification, the description of “substituted or (or) unsubstituted” means a fluorine atom; a chlorine atom; a bromine atom; a cyano group; a nitro group; a hydroxy group; A linear or branched alkyl group having 1 to 10 carbon atoms; an aryl group having 6 to 30 carbon atoms; a heteroaryl group having 2 to 30 carbon atoms; It is substituted with a substituent such as cycloalkyl group; or unsubstituted.

The molar ratio between p ₁ and q ₁ (p ₁ : q ₁ ) and the molar ratio between p ₂ and q ₂ (p ₂ : q ₂ ) is 1:50 to 1: 6,000, preferably 1 : 50-1: 4,000, more preferably 1: 100-1: 2,000. When the ratio of the fluorescent monomer compound is larger than the molar ratio 1:50, the degree of freedom is lost due to the bulkiness of the fluorescent monomer compound, and the interaction with the saccharide may be reduced. On the other hand, if the ratio of the fluorescent monomer compound is smaller than the molar ratio 1: 6,000, the absolute amount of fluorescence intensity may not be ensured. p ₁ and q ₁ are preferably about 1 to 100, and p ₂ and q ₂ are preferably about 50 to 600,000.

As described above, in the structure of the fluorescent hydrogel fiber of the present invention, the divalent organic residues Y ₁ and Y ₂ are preferably hydrophilic. Thereby, specifically, the following effects can be obtained. (1) Since the fluorescent monomer compound becomes water-soluble, the polymerization reaction when forming the fluorescent hydrogel fiber can be performed efficiently. (2) Introduction of a hydrophilic chain changes the environment and motility around phenylboronic acid that interacts with the substance to be detected, contributing to improved sensitivity, accuracy, response speed, and selectivity of the saccharide as the substance to be measured. . (3) The hydrophilic chain stabilizes the entire structure of the fluorescent hydrogel fiber. (4) Since the reaction can be performed only in water, the polymerization can be performed in a state suspended in an organic solvent.

  The weight average molecular weight of the fluorescent hydrogel fiber having the structure represented by the chemical formula 1 is preferably 50,000 to 750,000 in terms of polyethylene oxide by gel permeation chromatography (GPC), and preferably 150,000 to More preferably, it is 450,000.

  The shape of the fluorescent hydrogel fiber of the present invention is not particularly limited, and a cylindrical shape, a prismatic shape, a cubic shape, or a hollow shape thereof, or a three-dimensional structure constructed using these fibers, for example, a woven fabric structure, Any shape such as a cylinder structure, a tube, or a spring structure may be used. It is preferable to select a shape suitable for the method of embedding the fluorescent hydrogel fiber or fluorescence detection.

  The diameter of the fluorescent hydrogel fiber according to the present invention is not particularly limited, but is preferably 10 to 2000 μm and more preferably 100 to 1000 μm when embedding using a syringe, cannula or catheter. In this specification, a value measured with a stereomicroscope is used as the diameter.

  Moreover, it is preferable that the diameter of each fluorescent hydrogel fiber is substantially uniform so that each fluorescent hydrogel fiber has the same fluorescence intensity at a certain blood glucose level. In addition, when using two or more fluorescent hydrogel fibers of this invention, the diameter may mutually be the same and may differ.

  In addition, the length of the fluorescent hydrogel fiber of the present invention may be appropriately set according to the size (or length) of the site to be embedded in the body or the size (or length) of the installation site in the circulation path described later. There are no restrictions. As an example, if the ear is given as an implantation site, it is preferably in the range of 50 to 200 mm.

  The second of the present invention is a method for producing a fluorescent hydrogel fiber.

  The method for producing the fluorescent hydrogel fiber of the present invention is not particularly limited. For example, the method includes a step of producing an aqueous solution column using an aqueous solution containing a fluorescent monomer compound and a polymerizable monomer containing a (meth) acrylamide residue. A method is mentioned. The production method preferably further includes a step of polymerizing the aqueous solution column. Hereinafter, although such a manufacturing method is demonstrated in detail, this invention is not limited to this.

[(A) Step of producing aqueous solution column from aqueous solution]
The fluorescent monomer compound preferably has a structure represented by the following chemical formula 5.

In Formula 5, X ₁ , X ₂ , Z ₁ , Z ₂ , Y ₁ , Y ₂ , Q, Q ′, Q ″, and Q ′ ″ are the same definitions as in Formula 1.

Of these, 9,10-bis [[N- (2-boronobenzyl) -N- [6-[(acryloylpolyoxyethylene) carbonylamino] hexyl] amino] methyl]-, which is a preferable compound as the fluorescent monomer compound. 2-acetylanthracene (X ₁ and X _{2 in} Chemical Formula 5 are —C ₆ H ₁₂ —NHCO—, Y ₁ and Y ₂ are polyethylene glycol residues in which Chemical Formula 3 n is 2, Z ₁ and Z ₂ are -O-, a compound in which Q is an acetyl group, Q ′, Q ″, and Q ′ ″ are hydrogen atoms (hereinafter also simply referred to as “F-PEG-AAm”) is represented by the following reaction formula 1 Will be described with reference to FIG. However, the present invention is not limited to this.

Using 2-acetyl-9,10-dimethylanthracene (I in the above reaction formula 1) as a raw material, heating a carbon tetrachloride (CCl ₄ ) / chloroform mixed solvent, N-bromosuccinimide (NBS) and peroxide By reacting with benzoyl (BPO), 2-acetyl-9,10-bis (bromomethylene) anthracene (II in the above reaction scheme 1) is obtained. Then, this is reacted with N- (t-butoxycarbonyl) -hexyldiamine (III in the above reaction scheme 1 in the presence of a base such as diisopropylethylamine (DIEA) in a solvent such as N, N-dimethylformamide (DMF). ), The bromomethylene group becomes a [(t-butoxycarbonylamino) hexylamino] methylene group (IV in the above reaction scheme 1). When this is reacted with 2- (2-bromomethylphenyl) -1,3-dioxabonarine (V in the above reaction formula 1) in the presence of a base such as DIEA in a solvent such as DMF, 9,10- Bis [[N-6 ′-(t-butoxycarbonylamino) hexyl-N- [2- (5,5-dimethylborinan-2-yl) benzyl] amino] methyl] -2-acetylanthracene (the above reaction formula VI) in 1 is obtained. When this was deprotected by the action of an acid such as hydrochloric acid, 9,10-bis [[N- (6′-aminohexyl) -N- (2-boronobenzyl) amino] methyl] -2-acetylanthracene (above VII) in scheme 1 is obtained. Next, when acryloyl- (polyethylene glycol) -N-hydroxysuccinimide ester is reacted in a basic buffer, F-PEG-AAm, which is the target product, can be obtained.

  When a compound having an acyl group other than an acetyl group in the anthracene skeleton is used as the raw material compound, the anthracene skeleton other than the acetyl group can be selected by appropriately selecting a solvent, an additive, a reaction temperature, a reaction time, a separation method, and the like. A compound having an acyl group can be produced.

  As the polymerizable monomer containing the (meth) acrylamide residue, the obtained polymer may have a (meth) acryloyl group and an amide in its structure, and (meth) acrylamide or a derivative thereof is preferable. Specific examples include, for example, acrylamide, methacrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-tert-butylacrylamide, N-tris-hydroxymethylacrylamide, N-hydroxymethylacrylamide, or N- (N-butoxymethyl) acrylamide and the like can be mentioned. Moreover, the condensate of (meth) acryloyl chloride, such as N-acryloyl lysine and N-acryloyl hexamethylene diamine, and the compound which has an amino acid or an active amino group can also be used. More preferred is acrylamide or methacrylamide.

  In this step, the polymerizable monomer containing the fluorescent monomer compound and the (meth) acrylamide residue is in the form of an aqueous solution.

  The solvent of the aqueous solution is not particularly limited, and distilled water, ion exchange water, pure water, ultrapure water, or the like can be used. In addition, an aqueous solution in which a fluorescent monomer compound and a polymerizable monomer having a (meth) acrylamide residue are added to a phosphate buffer can also be used.

  The concentration of the fluorescent monomer compound in the aqueous solution is preferably 1 to 30% by mass, and more preferably 2 to 10% by mass. Moreover, it is preferable that the density | concentration in the aqueous solution of the polymerizable monomer containing the said (meth) acrylamide residue is 5-50 mass%, and it is more preferable that it is 10-20 mass%.

  The aqueous solution may contain other components. Examples of such components include a polymerization initiator, a polymerization accelerator, a crosslinking agent, a cationic monomer that can be a cation in water, an anionic monomer that can be an anion in water, or a nonionic monomer having no ion. Is mentioned. Details of the polymerization initiator, the polymerization accelerator, and the crosslinking agent will be described later.

  Examples of the cationic monomer that can be a cation in water include dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, and 4-vinylpyridine. These may be used alone or in combination of two or more.

  Examples of anionic monomers that can be anions in water include (meth) acrylic acid, vinylpropionic acid, 4-vinylbenzenesulfonic acid, and the like. These may be used alone or in combination of two or more.

  Examples of nonionic monomers having no ions include, for example, 2-hydroxyethyl (meth) acrylate, 3-methoxypropyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate, 2-methoxyethyl acrylate, or 1, Examples include 4-cyclohexanedimethanol monoacrylate. These may be used alone or in combination of two or more.

  The blending amount of these other components is preferably from 0.1 to 10 mol%, more preferably from 2 to 7 mol%, based on the total amount of the fluorescent monomer compound and the polymerizable monomer containing a (meth) acrylamide residue. is there.

  Moreover, it is preferable that the density | concentration in the aqueous solution of these other components is 0.01-10 mass%, and it is more preferable that it is 0.02-1.0 mass%. Details of the polymerization initiator, the polymerization accelerator, and the crosslinking agent will be described later.

  Examples of a method for producing an aqueous solution column from an aqueous solution containing the fluorescent monomer compound and the polymerizable monomer containing the (meth) acrylamide residue include (1) a method using a tube; (2) a method using a microchannel. And (3) a method using a coaxial microfluidic device.

  The method using the tube of (1) is not particularly limited. For example, an aqueous solution containing a fluorescent monomer compound and a polymerizable monomer containing a (meth) acrylamide residue is injected into the tube to produce an aqueous solution column. The method of doing is mentioned.

  FIG. 1 is a schematic diagram showing an aqueous solution column 12 filled in a tube 11. The aqueous solution column 12 is polymerized to become a fluorescent hydrogel fiber, and the fluorescent hydrogel fiber can be obtained by taking it out from the tube 11.

  Examples of the material of the tube include glass, metal, such as aluminum, steel, and plastic, such as polyolefin, silicon, and Teflon.

  The aqueous solution can be injected into the tube by using a syringe or the like to obtain an aqueous solution column. After injecting the aqueous solution into the tube, plug both ends of the tube as necessary. A fluorescent hydrogel fiber can be obtained by polymerizing the aqueous solution column thus prepared by various polymerization methods. In the method using a tube, fluorescent hydrogel fibers having various shapes in which the cross section of the fiber and the shape in the length direction are changed can be produced by changing the shape of the tube. Details of various polymerization methods will be described later.

  The inner diameter and length of the tube can be appropriately set depending on the size of the target fluorescent hydrogel fiber. For example, the inner diameter of the tube is preferably 10 to 2000 μm, and the length of the tube is preferably 10 to 300 mm.

  The fluorescent hydrogel fiber polymerized in the tube can be taken out by extruding with pressure or the like, shrinking the fluorescent hydrogel fiber, cutting the tube, or dissolving the tube. When the tube is extruded and taken out, it can be easily taken out by coating the inner wall of the tube with a surfactant, for example, Pluronic (registered trademark), polyethylene glycol, MPC polymer or the like.

  An example of a microchannel (microchannel) used in the method (2) using the microchannel (microchannel) is shown in FIG. 2A. When an aqueous solution containing a fluorescent monomer compound and a polymerizable monomer containing a (meth) acrylamide residue is injected into the inlet 21 of the microchannel (microchannel), it flows to the outlet 22 and becomes a microchannel (microchannel) 23. The aqueous solution column 24 can be produced therein. FIG. 2B is a schematic diagram showing a cross section taken along the line AA in FIG. 2A, in a space sandwiched between a substrate 26 including a microchannel (microchannel) 23 and a substrate 27 that covers the top of the substrate 26. It is shown that the aqueous solution 24 is formed by injecting the aqueous solution. Examples of the material for forming the micro flow path (micro flow path) include glass, polydimethylsiloxane, acrylic resin, polymethyl methacrylate resin, and the like.

  A fluorescent hydrogel fiber can be obtained by polymerizing the aqueous solution column thus prepared by various polymerization methods. Details of various polymerization methods will be described later. In the method using a microchannel (microchannel), fluorescent hydrogel fibers having various shapes in which the cross section of the fiber and the shape in the length direction are changed can be produced by changing the shape of the channel.

  The inner diameter and length of the microchannel (microchannel) can be appropriately set depending on the size of the target fluorescent hydrogel fiber. For example, the inner diameter of the microchannel (microchannel) is preferably 10 to 2000 μm, and the length of the microchannel (microchannel) is preferably 10 to 3000 mm.

The fluorescent hydrogel fiber obtained by polymerization in the microchannel (microchannel) can be taken out from the substrate 26 including the microchannel (microchannel) 23 by removing the substrate 27 placed thereon. When removing, pre-coated inner wall of the microchannel (fine flow path) surfactants such as Pluronic (registered trademark), a polymer such as polytetrafluoroethylene or octafluorocyclobutane _{(Octafluorocyclobutane,} C 4 _{F 8)} or the like, It can be easily taken out.

  In the method (3) using the coaxial microfluidic device, for example, a coaxial microfluidic device as shown in FIGS. 3A and 3B can be used. A microfluidic device that can be injected separately into a core portion and a shell portion so that two fluids are coaxial is disclosed in, for example, Lab Chip, 4, pp. 576-580, 2004, FIG. 1 is described in detail. For example, by using an aqueous solution containing a fluorescent monomer compound and a polymerizable monomer containing a (meth) acrylamide residue as the core fluid 31, and using an organic solvent that is not mixed with the aqueous solution as the shell fluid 32, the shell The aqueous column of the core part can be prepared in the organic solvent of the part. Examples of the organic solvent include cyclohexane, liquid paraffin, hexadecane, corn oil, mineral oil, silicone oil and the like, and these can be used alone or in combination of two or more. By polymerizing this aqueous solution column by various polymerization methods, the fluorescent hydrogel fiber of the present invention can be obtained. Moreover, you may use the solution which has a composition which superposes | polymerizes the aqueous solution of a core part instantaneously as the fluid 32 of a shell part instead of the said organic solvent by contacting with a core part. Details of various polymerization methods will be described later.

[(B) Step of polymerizing aqueous solution column to produce fluorescent hydrogel fiber]
The fluorescent hydrogel fiber of the present invention can be produced by polymerizing the aqueous solution column obtained by the above method (1), (2), or (3). The method for polymerizing the aqueous solution column is not particularly limited, and examples thereof include a chemical polymerization method using a radical polymerization initiator, a photopolymerization method using a photopolymerization initiator, or a radiation polymerization method of irradiating radiation.

  The chemical polymerization method is performed by adding a radical polymerization initiator and, if necessary, a polymerization accelerator to an aqueous solution, an organic solvent, or both. The polymerization temperature is preferably 15 to 75 ° C, more preferably 20 to 60 ° C. The polymerization time is preferably 3 minutes to 20 hours, more preferably 10 minutes to 8 hours. Examples of radical polymerization initiators include, for example, persulfates such as sodium persulfate, potassium persulfate, or ammonium persulfate; hydrogen peroxide; azo such as azobis-2-methylpropionamidine hydrochloride or azoisobutyronitrile. Compound: Peroxides such as benzoyl peroxide, lauroyl peroxide, cumene hydroperoxide or benzoyl oxide can be used, and these can be used alone or in combination of two or more. In this case, as a polymerization accelerator, for example, a reducing agent such as sodium hydrogen sulfite, sodium sulfite, Mohr's salt, sodium pyrobisulfite, sodium formaldehyde sulfoxylate, or ascorbic acid; ethylenediamine, sodium ethylenediaminetetraacetate, glycine, or N , N, N ′, N′-Amine compounds such as tetramethylethylenediamine;

  The amount of the polymerization initiator is preferably 0.01 to 10% by mass as the concentration in the aqueous solution column.

  The photopolymerization method can be performed, for example, by adding a photopolymerization initiator to an aqueous solution in advance and irradiating an aqueous solution column obtained in an organic solvent with ultraviolet light.

  Examples of the photopolymerization initiator used include, for example, acetophenone, 2,2-diethoxyacetophenone, p-dimethylaminoacetophenone, p-dimethylaminopropiophenone, p-dimethylaminopropiophenone, benzophenone, p, p '-Dichlorobenzophenone, p, p'-bisdiethylaminobenzophenone, Michler's ketone, benzyl, benzoin, benzoin methyl ether, benzoin isopropyl ether, benzoin n-propyl ether, benzoin isobutyl ether, benzyl dimethyl ketal, 1-hydroxy-cyclohexyl phenyl ketone , Tetramethylthiuram monosulfide, thioxanthone, 2-chlorothioxanthone, 2,4-dimethylthioxanthone, 2,2-dimethylpropioyl Phenylphosphine oxide, 2-methyl-2-ethylhexanoyldiphenylphosphine oxide, 2,6-dimethylbenzoyldiphenylphosphine oxide, 2,6-dimethoxybenzoyldiphenylphosphine oxide, 2,4,6-trimethylbenzoyldiphenyl Phosphine oxide, bis (2,6-dimethoxybenzoyl) -2,4,4-trimethylpentylphosphine oxide, 2,3,6-trimethylbenzoyl-diphenylphosphine oxide, bis (2,3,6-trimethylbenzoyl) ) -Phenylphosphine oxide, 2,4,6-trimethoxybenzoyl-diphenylphosphine oxide, 2,4,6-trichlorobenzoyldiphenylphosphine oxide, 2,4 6-trimethylbenzoylnaphthyl phosphonate, p-dimethylaminobenzoic acid, p-diethylaminobenzoic acid, azobisisobutyronitrile, 1,1′-azobis (1-acetoxy-1-phenylethane), benzoin peroxide, Examples include di-tert-butyl peroxide. These may be used alone or in combination of two or more.

Wavelength is preferably 200~400nm of ultraviolet light, The irradiation amount of ultraviolet light is preferably 100 to 2000 mJ / cm ^2, more preferably 500~1500mJ / cm ^2.

  In the radiation polymerization method, radiation polymerization is performed by irradiating the obtained aqueous solution column with radiation. The radiation is preferably an electron beam, and the irradiation dose is preferably 10 to 200 kGy, more preferably 20 to 50 kGy.

  When the polymerization is carried out with an electron beam, the polymerization is possible without using a polymerization initiator or a polymerization accelerator, and in that case, the step of washing the polymerization initiator / accelerator need not be performed. Moreover, the polymerization initiator and the polymerization accelerator which were illustrated in the said chemical polymerization method can also be used.

  The electron beam irradiation can be performed by an electron accelerator. The apparatus is classified into a low energy type, a medium energy type, and a high energy type according to the magnitude of the voltage for accelerating electrons. In the polymerization in this step, a low energy type electron beam accelerator is preferable. For example, as an example of a low energy type electron beam accelerator, a low energy electron beam irradiation device manufactured by Hamamatsu Photonics Co., Ltd. may be mentioned. This is to accelerate the thermoelectrons generated from the filament at a high voltage to increase the energy and take out the electron beam from the beryllium window foil into the atmosphere, and irradiate the electron beam with a relatively low acceleration voltage of 40 to 110 kV. Is possible.

  After the fluorescent hydrogel fiber is obtained by the above process, it is preferable to further wash the fluorescent hydrogel fiber using a cleaning liquid. Examples of the cleaning liquid include a phosphate buffer and pure water.

  In addition, when an organic solvent is used in the step (a), the dispersion of the fluorescent hydrogel fiber is sequentially performed using a cleaning liquid A that can dissolve the organic solvent, a cleaning liquid B that can dissolve the cleaning liquid A, and the like. By exchanging and washing the phases, a fluorescent hydrogel fiber can be finally obtained in an aqueous solution. Examples of the cleaning liquid include, for example, alcohols, ethers, esters, ketones, hydrocarbons, halogen-containing hydrocarbons, buffer solution, pure water, etc., preferably methanol, ethanol, propanol, Examples include butanol, diethyl ether, ethyl acetate, acetone, heptane, hexane, cyclohexane, chloroform, methylene chloride, phosphate buffer, pure water, and more preferably hexane, ethanol, phosphate buffer, pure water, and the like. Can be mentioned. These cleaning solutions can be used alone or in combination of two or more. Among these, at least one selected from the group consisting of hexane, ethanol, phosphate buffer, and pure water is preferable.

  The fluorescent hydrogel fiber obtained by the production method of the present invention as described above preferably has a structure represented by the above chemical formula 1. Since the detailed structure is the same as the above-mentioned content, description is abbreviate | omitted here.

  The fluorescent hydrogel fiber having the structure represented by the chemical formula 1 is produced without copolymerization of the fluorescent monomer compound represented by the chemical formula 5 and a polymerizable monomer containing a (meth) acrylamide residue. Is possible. For example, an aqueous solution containing a polymer obtained by polymerizing a polymerizable monomer having a (meth) acrylamide residue in advance, a fluorescent monomer compound represented by the above chemical formula 5, a polymerization initiator, and, if necessary, a polymerization accelerator. A fluorescent hydrogel fiber having the structure represented by the above chemical formula 1 can also be produced by adding an aqueous solution column to an organic solvent and polymerizing the obtained aqueous solution column by the above polymerization method.

According to the production method of the present invention, the fluorescent hydrogel fiber may have a three-dimensional crosslinked structure. The method for introducing the three-dimensional crosslinked structure is not particularly limited, and includes, for example, a fluorescent monomer compound represented by the above chemical formula 1 and a (meth) acrylamide residue by allowing a crosslinking agent to act on the fluorescent hydrogel fiber of the present invention. A method of forming intermolecular crosslinks at least partly with the polymerizable monomer can be mentioned. When a three-dimensional crosslink is formed on a poly (meth) acrylamide chain, saccharides can be easily detected without eluting the fluorescent monomer compound even in an aqueous solution. The fluorescent monomer compound used in the present invention has a hydrophobic moiety that emits fluorescence by binding to a saccharide, and the hydrophobic moiety is a divalent compound represented by Y ₁ and Y ₂ in the above chemical formula 1. Since it is bonded to a poly (meth) acrylamide chain via an organic residue, a degree of freedom for bonding with a saccharide is ensured even in an aqueous solution. Therefore, even if a three-dimensional crosslinked structure is formed, the detection sensitivity of saccharides is hardly lowered.

  Examples of the crosslinking agent widely include those capable of introducing a three-dimensional crosslinked structure into a fluorescent sensor compound by a polymerizable double bond. Specific examples include, for example, N, N′-methylenebis (meth) acrylamide, N, N ′-(1,2-dihydroxyethylene) -bis (meth) acrylamide, diethylene glycol di (meth) acrylate, (poly) Ethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, propylene glycol di (meth) acrylate, trimethylolpropane di (meth) acrylate, trimethylolpropane tri (meth) acrylate, pentaerythritol di (meth) acrylate , Pentaerythritol tri (meth) acrylate, pentaerythritol tetra (meth) acrylate, (poly) propylene glycol di (meth) acrylate, glycerol tri (meth) acrylate, glycerol acrylic Divinyl compounds such as thiomethacrylate, ethylene oxide modified trimethylolpropane tri (meth) acrylate, pentaerythritol tetra (meth) acrylate, dipentaerythritol hexa (meth) acrylate; triallyl cyanurate, triallyl isocyanurate, triallyl phosphate , Triallylamine, poly (meth) allyloxyalkane, (poly) ethylene glycol diglycidyl ether, glycerol diglycidyl ether, ethylene glycol, polyethylene glycol, propylene glycol, glycerin, pentaerythritol, ethylenediamine, polyethyleneimine, glycidyl (meta ) Acrylate, triallyl isocyanurate, trimethylolpropane di (meth) allyl ether, Tet Arirokishietan or such as glycerol propoxy triacrylate. These crosslinking agents can be used alone or in combination of two or more.

  The amount of the crosslinking agent is preferably 0.01 to 10% by mass as the concentration in the aqueous solution column.

  The fluorescent hydrogel fiber obtained by the present invention can be used as a component of a saccharide measuring sensor for implantation in the body. The fluorescent hydrogel fiber of the present invention can be implanted in a living body with minimal invasiveness. Examples of the embedding method include embedding using a syringe, cannula, indwelling needle, or catheter, or embedding by removing a part of the skin surface layer. By embedding the fluorescent hydrogel fiber embedded and pulling it out, all the fluorescent hydrogel fibers embedded in the living body can be taken out. That is, the present invention also provides a method for using such a fluorescent hydrogel fiber.

  Since the fluorescent hydrogel fiber of the present invention has a poly (meth) acrylamide structure with high biocompatibility, there is little influence on the living body during long-term implantation in the living body.

  Since the fluorescent hydrogel fiber of the present invention is not subject to chemical degradation such as hydrolysis or enzymatic degradation in vivo, it is considered that the stability of long-term implantation is high. The embedded tissue is preferably intradermal or subcutaneous, and in order to achieve high sensitivity and a small time lag, a dermis layer that actively exchanges body fluids with blood and has a shallow depth from the skin surface is more preferable. The implantation place is not particularly limited as long as it can be optically measured from the skin surface, and examples thereof include arms, legs, abdomen, and ears. In addition, fluorescent hydrogel fibers can be fixed in the body by sewing or wrapping the tissue under the skin or inside the body, and can be implanted without being limited to the size or shape of the part or organ. Can do. For example, it can be fixed by sewing into a subcutaneous tissue, kidney capsule, pancreas capsule or the like, or wrapping around a blood vessel or the like.

  The fluorescent hydrogel fiber of the present invention is embedded in the body, and has means for measuring the fluorescence generated from the fluorescent hydrogel fiber by irradiating excitation light from a sensor embedded outside or in the same site. It can also be used as a component of a saccharide measurement sensor.

  Further, the body fluid is circulated between the hollow needle inserted into the body and the fluorescence detection system outside the body connected to the hollow needle, and a fluorescent hydrogel fiber is attached to a part of the circulation path, and the fluorescence hydro It can also be used as a component of a saccharide measurement sensor having means for measuring fluorescence generated from gel fibers.

  The circulation path preferably has a property of allowing saccharides to be measured to permeate but not allowing permeation of macromolecules such as cells and proteins among biological components. The body fluid passes through the circulation path to which the fluorescent hydrogel fiber is attached, and sugars in the body fluid come into contact with the fluorescent hydrogel fiber. By continuously measuring the fluorescence of the fluorescent hydrogel fiber in contact with the saccharide in the body fluid using a fluorescence detection system, a value reflecting the sugar concentration in the living body can be obtained.

  The fluorescence detection system preferably includes at least one light source and at least one photodetector. The light source and the light detector are preferably arranged so that excitation light emitted from the light source does not enter as much as possible, and fluorescence emitted from the fluorescent hydrogel fiber can be detected efficiently.

  For fluorescent signal measurement, lamps, LEDs, lasers, and other light sources and photodetectors that match the fluorescence characteristics of the fluorescent hydrogel fiber, and if necessary, optical fiber, lens, mirror, prism, optical filter, etc. are configured as sensors. It can be carried out by arranging it at an appropriate position around the object.

  The saccharide that can be detected using the fluorescent hydrogel fiber of the present invention is not particularly limited, and examples thereof include monosaccharides such as glucose, galactose, and fructose, and polysaccharides such as maltose, sucrose, and lactose. Among these, glucose is more preferable.

  By using the above-mentioned saccharide measurement sensor, it is possible to reduce the complexity or time lag of the blood glucose level when a diabetic patient self-controls the blood glucose level. Furthermore, by using the saccharide measurement sensor described above, it is possible for people other than diabetics to easily perform blood glucose measurement for health care.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, these Examples do not restrict | limit this invention at all.

Example 1: Synthesis of 9,10-bis [[N- (2-boronobenzyl) -N- [6-[(acryloylpolyoxyethylene) carbonylamino] hexyl] amino] methyl] -2-acetylanthracene)
(A) Synthesis of 9,10-bis (bromomethyl) -2-acetylanthracene (II in the above reaction scheme 1) 9,10-dimethyl-2-acetylanthracene (I in the above reaction scheme 1) 600 mg, N- Bromosuccinimide (800 mg) and benzoyl peroxide (5 mg) were added to a mixture of chloroform (6 mL) and carbon tetrachloride (20 mL), and the mixture was heated to reflux at 80 ° C. for 2 hours. After removing the solvent, the residue was extracted with methanol to obtain 780 mg of the desired product.

(B) Synthesis of 9,10-bis [6 ′-(t-butoxycarbonylamino) hexylaminomethyl] -2-acetylanthracene (IV in Reaction Scheme 1) 500 mg of the product obtained in (A) above, 1.125 g of N-BOC-hexyldiamine (III in the above reaction scheme 1) and 1.25 mL of diisopropylethylamine are dissolved in 10 mL of N, N-dimethylformamide (DMF), and stirred at 45 ° C. for 1 hour to react. Went. The reaction mixture was diluted with 60 mL of chloroform, washed 3 times with 100 mL of water and once with 100 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The desiccant was filtered, concentrated, and purified by silica gel column chromatography using a mixed solvent of chloroform / methanol (volume ratio 95/5) as an eluent to obtain 367 mg of the desired product.

(C) 9,10-bis [[N-6 ′-(t-butoxycarbonylamino) hexyl-N- [2- (5,5-dimethylborinan-2-yl) benzyl] amino] methyl] -2 -Synthesis of acetylanthracene (VI in the above reaction scheme 1) 200 mg of the product obtained in the above (B), 700 mg of 2- (2-bromomethylphenyl) -1,3-dioxaborinane (V in the above reaction scheme 1) And 0.35 mL of N, N-diisopropylethylamine was dissolved in 3 mL of dimethylformamide and stirred at room temperature (25 ° C.) for 16 hours. After removing the solvent, the residue was purified by a silica gel column using methanol / chloroform as an eluent to obtain 194 mg of the desired product.

(D) Synthesis of 9,10-bis [[N- (6′-aminohexyl) -N- (2-boronobenzyl) amino] methyl] -2-acetylanthracene (VII in the above reaction scheme 1) ) Was dissolved in 2 mL of methanol, 2 mL of 4N hydrochloric acid was added, and the mixture was stirred at room temperature (25 ° C.) for 10 hours. After evaporation to dryness, inorganic salts were removed by gel filtration to obtain 95 mg of the desired product.

(E) Synthesis of F-PEG-AAm 160 mg of the product obtained in (D) above was dissolved in 0.5 mL of DMF, and acryloyl- (polyethylene glycol) -N-hydroxysuccinimide ester (polyethylene glycol residue part) Was added to a solution of 1.22 g of 10 mL 100 mM phosphate buffer (pH = 8.0) and stirred at room temperature (25 ° C.) for 20 hours. The reaction mixture was subjected to gel filtration, and the fluorescent polymer fraction was collected. After lyophilization, 1.2 g of the desired product, F-PEG-AAm, was obtained. The ¹ H-NMR data measured using deuterated chloroform is as shown in Table 1 below, and no hydrogen signal overlapping a peak derived from a polyethylene glycol (PEG) residue was confirmed.

(Example 2: Production of fluorescent hydrogel fiber)
Acrylamide and N, N′-methylenebisacrylamide were added to a 60 mM phosphate buffer (final concentration of 15% by weight of acrylamide and 0.3% by weight of N, N′-methylenebisacrylamide). Each was added to pH 7.4) and dissolved and mixed to prepare an aqueous solution. Next, nitrogen bubbling was performed on the aqueous solution at room temperature (25 ° C.).

  In the aqueous solution, the concentration of F-PEG-AAm synthesized in Example 1 was 5% by mass, the concentration of sodium persulfate was 0.9% by mass, and N, N, N ′, N′-tetramethylethylenediamine was 0. Each was added to a concentration of 2% by mass.

Next, the aqueous solution was sucked into a polyolefin tube coated with Pluronic (registered trademark) to facilitate removal of the produced fluorescent hydrogel fiber, and polymerization was started at room temperature (25 ° C.) in a nitrogen atmosphere. . The polyolefin tube has an inner diameter of 400 μm and a length of 150 mm, an inner diameter of 700 μm and a length of 150 mm,
A total of 3 pieces each having an inner diameter of 1000 μm and a length of 150 mm were used.

  After 10 minutes, air pressure was applied, and the fluorescent hydrogel fiber obtained by polymerization was taken out from the polyolefin tube. By using polyolefin tubes having different inner diameters, three fluorescent hydrogel fibers each having a diameter of 400 μm, 700 μm, and 1000 μm and a length of about 150 mm could be obtained. FIGS. 4 and 5 show photographs of the obtained fluorescent hydrogel fiber having a diameter of 700 μm observed with a fluorescence microscope. 4 is a photograph of a bright field image, and FIG. 5 is a photograph of a fluorescence image.

  The fluorescent hydrogel fiber having a diameter of 700 μm obtained in this example was immersed in a 60 mM phosphate buffer prepared so that the glucose concentration was in the range of 0 to 1000 mg / dL, and each glucose concentration was measured with a fluorescent stereomicroscope. The fluorescence intensity of the fluorescent hydrogel fiber was observed. The results are shown in FIG.

  As shown in FIG. 6, the fluorescent hydrogel fiber has glucose responsiveness, and it was confirmed that the glucose concentration can be measured by using the fluorescent hydrogel fiber of the present invention.

(Example 3: Confirmation of glucose responsiveness in vivo)
When the glucose solution was administered from the abdominal cavity of the mouse to increase the blood glucose level of the mouse from 114 mg / dL to 600 mg / dL or more, the change in the fluorescence intensity in the body of the fluorescent hydrogel fiber was observed. The fluorescent hydrogel fiber having a diameter of 700 μm obtained in Example 2 was cut into a length of about 50 mm and used. Photographs of mouse ears embedded with fluorescent hydrogel fibers are shown in FIGS. FIG. 7 is a photograph of a bright field image, and FIG. 8 is a photograph of a fluorescence image.

  FIG. 9 is a photograph of a fluorescence image obtained by observing the ear of a mouse with a fluorescence microscope when the blood glucose level was 114 mg / dL, the blood glucose level was 383 mg / dL, and the blood glucose level was 600 mg / dL or higher. FIG. 10 is a color image representing the fluorescence intensity obtained by analyzing the photograph of the fluorescence image of FIG. As shown in FIG. 9 and FIG. 10, it can be confirmed that the fluorescence intensity of the fluorescent hydrogel fiber increases as the blood glucose level increases, and the fluorescent hydrogel fiber of the present invention can be used as a saccharide measuring sensor for implantation in the body. It could be confirmed.

(Example 4: Production of device for embedding fluorescent hydrogel fiber)
A device for easily embedding the fluorescent hydrogel fiber in the body with minimal invasiveness was produced by improving the indwelling needle as shown in FIG. The tip of the inner needle 111 of the indwelling needle was shaved and blunted (reference numeral 112), and the inner needle 111 was filled with PDMS (polydimethylsiloxane, reference numeral 113). Further, the fluorescent hydrogel fiber 115 obtained in Example 2 was packed in the outer needle 114 to complete the device for implantation.

(Example 5: Embedding / removing of fluorescent hydrogel fiber and confirmation of responsiveness in vivo)
In this example, in order to observe the fluorescence of the fluorescent hydrogel fiber embedded in the living body from the outside, the ears of mice with thin skin were targeted for implantation.

  As shown in the schematic diagram of FIG. 12, among the implantable devices produced in Example 4, the inner needle 111 blunted so that the ears of the mouse were not broken was inserted into the ears 116 of the mouse ( In FIG. 12A, the inner needle 111 was pulled out to create a space 117 for embedding a fiber in the mouse ear 116 (FIG. 12B). Next, after inserting the outer needle 114 of the indwelling needle containing the fluorescent hydrogel fiber 115 into the space prepared above, the inner needle of the indwelling needle was inserted into the outer needle (C in FIG. 12). By pushing the inner needle 111 and finally pulling out the outer needle together with the inner needle, the fluorescent hydrogel fiber 115 could be embedded in the mouse ear 116 (D in FIG. 12).

  A photograph of a mouse ear embedded with a fluorescent hydrogel fiber by this method observed with a fluorescent microscope is shown in FIGS. FIG. 13 is a photograph of a bright field image, and FIG. 14 is a photograph of a fluorescence image.

  Further, the fluorescent hydrogel fiber was not only easily implanted with minimal invasiveness, but also could be easily removed from the body one day after implantation by grasping and pulling the end of the fluorescent hydrogel fiber. 15 and 16 show observation images of the mouse ears after the embedded fluorescent hydrogel fiber is taken out. FIG. 15 is a photograph of a bright field image, and 16 is a photograph of a fluorescent image. FIG. 17 shows a photograph of the fluorescent hydrogel fiber taken out from the mouse ear.

11 tubes,
12, 24 Aqueous solution column,
21 Microchannel (microchannel) inlet,
22 Outlet of microchannel (microchannel),
23 microchannel (microchannel),
26, 27 substrates,
31 core fluid,
32 Shell fluid,
33 Glass tube for injection,
34 Polydimethylsiloxane holder,
35 Glass tube for focusing,
36, 115 fluorescent hydrogel fiber,
37 DI water,
111 Inner needle,
112 The tip of the inner needle,
113 polydimethylsiloxane,
114 outer needle,
116 mouse ears,
117 space.

Claims (11)

A fluorescent hydrogel fiber having a structure represented by the following chemical formula 1:
In the chemical formula 1,
X ₁ and X ₂ may be the same or different and are —COO—, —OCO—, —CH ₂ NR—, —NR—, —NRCO—, —CONR—, —SO ₂ NR—, —NRSO ₂ —. , -O-, -S-, -SS-, -NRCOO-, -OCONR-, and -CO-, an alkylene group having 1 to 30 carbon atoms and containing at least one substituent selected from the group consisting of In this case, R is a hydrogen atom or a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms,
Z ₁ and Z ₂ may be the same or different and are —O— or —NR′—, wherein R ′ is a hydrogen atom or a substituted or unsubstituted straight chain having 1 to 10 carbon atoms. A branched or branched alkyl group,
Q, Q ′, Q ″ and Q ′ ″ may be the same or different, and are a hydrogen atom, a hydroxy group, a substituted or unsubstituted C 1-10 linear or branched An alkyl group, an acyl group having 2 to 11 carbon atoms, a substituted or unsubstituted linear or branched alkoxy group having 1 to 10 carbon atoms, a group containing a halogen atom, a carboxyl group, a carboxylic acid ester group , A carboxylic acid amide group, a cyano group, a nitro group, an amino group, or an alkylamino group having 1 to 10 carbon atoms, and at least one of Q and Q ′ and Q ″ and Q ′ ″ is bonded to each other May form an aromatic ring or a heterocyclic ring,
Y ₁ and Y ₂ may be the same or different and may be substituted divalent organic residues. At this time, at least one of Y ₁ and Y ₂ is represented by the following chemical formula 3 or the following chemical formula 4 It may contain the structure represented by
In the chemical formula 3 and the chemical formula 4, n is 2 to 5, j is 1 to 5, m is 1 to 200, * represents a bonding point,
A ₁ and A ₂ may be the same or different and each is a hydrogen atom or a methyl group;
U ₁ , U ₂ , U ₃ and U ₄ may be the same or different and are a hydrogen atom or a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms. ,
The molar ratio of p ₁ and q ₁ (p ₁ : q ₁ ) and the molar ratio of p ₂ and q ₂ (p ₂ : q ₂ ) are 1:50 to 1: 6,000.   The fluorescent hydrogel fiber according to claim 1, which has a three-dimensional crosslinked structure.   The fluorescent hydrogel fiber according to claim 1 or 2, having a diameter of 10 to 2000 µm. (A) A method for producing a fluorescent hydrogel fiber, comprising a step of producing an aqueous solution column from an aqueous solution containing a fluorescent monomer represented by the following chemical formula 5 and a polymerizable monomer containing a (meth) acrylamide residue:
In Formula 5,
X ₁ and X ₂ may be the same or different and are —COO—, —OCO—, —CH ₂ NR—, —NR—, —NRCO—, —CONR—, —SO ₂ NR—, —NRSO _2. C1-C30 linear containing at least one substituent selected from the group consisting of-, -O-, -S-, -SS-, -NRCOO-, -OCONR-, and -CO-. Or a branched alkylene group, wherein R is a hydrogen atom, or a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms;
Z ₁ and Z ₂ may be the same or different and are —O— or —NR′—, wherein R ′ is a hydrogen atom or a substituted or unsubstituted straight chain having 1 to 10 carbon atoms. A branched or branched alkyl group,
Q, Q ′, Q ″ and Q ′ ″ may be the same or different, and are a hydrogen atom, a hydroxy group, a substituted or unsubstituted C 1-10 linear or branched An alkyl group, an acyl group having 2 to 11 carbon atoms, a substituted or unsubstituted linear or branched alkoxy group having 1 to 10 carbon atoms, a group containing a halogen atom, a carboxyl group, a carboxylic acid ester group , A carboxylic acid amide group, a cyano group, a nitro group, an amino group, or an alkylamino group having 1 to 10 carbon atoms, and at least one of Q and Q ′ and Q ″ and Q ′ ″ is bonded to each other May form an aromatic ring or a heterocyclic ring,
Y ₁ and Y ₂ may be the same or different and may be substituted divalent organic residues. At this time, at least one of Y ₁ and Y ₂ is represented by the following chemical formula 3 or the following chemical formula 4 It may contain the structure represented by
In Chemical Formula 3 and Chemical Formula 4, n is 2 to 5, j is 1 to 5, m is 1 to 200, and * represents a bonding point.   Furthermore, the manufacturing method of the fluorescent hydrogel fiber of Claim 4 including the process of producing the fluorescent hydrogel fiber by polymerizing the said aqueous solution column (b).   The said (a) process is a manufacturing method of the fluorescent hydrogel fiber of Claim 4 or 5 using a tube, a microchannel, or a coaxial microfluidic device.   In the step (b), the aqueous solution column is chemically polymerized using a radical polymerization initiator, the aqueous solution column is photopolymerized using a photopolymerization initiator, or the aqueous solution column is irradiated with radiation to form the aqueous solution column. The method for producing a fluorescent hydrogel fiber according to claim 5 or 6, wherein the fluorescent hydrogel fiber is subjected to radiation polymerization.   Saccharide measurement for implantation in the body, comprising the fluorescent hydrogel fiber according to any one of claims 1 to 3 or the fluorescent hydrogel fiber obtained by the production method according to any one of claims 4 to 7. Sensor.   The fluorescent hydrogel fiber according to any one of claims 1 to 3 or the fluorescent hydrogel fiber obtained by the production method according to any one of claims 4 to 7 is implanted in the body, from inside or outside the body. A saccharide-measuring sensor for implantation in the body, comprising means for irradiating excitation light and measuring fluorescence generated from the fluorescent hydrogel fiber.   The fluorescent hydrogel fiber according to any one of claims 1 to 3, or the fluorescent hydrogel fiber obtained by the production method according to any one of claims 4 to 7, at least in a path through which a body fluid circulates. A saccharide measurement sensor having means for attaching to a part and measuring fluorescence generated from the fluorescent hydrogel fiber.   The usage method of the fluorescent hydrogel fiber of any one of Claims 1-3, or the fluorescent hydrogel fiber obtained by the manufacturing method of any one of Claims 4-7.