JP4691333B2 - Fluorescent monomer compound for saccharide measurement, fluorescent sensor substance for saccharide measurement, and saccharide measurement sensor for implantation in the body - Google Patents
Fluorescent monomer compound for saccharide measurement, fluorescent sensor substance for saccharide measurement, and saccharide measurement sensor for implantation in the body Download PDFInfo
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- JP4691333B2 JP4691333B2 JP2004216535A JP2004216535A JP4691333B2 JP 4691333 B2 JP4691333 B2 JP 4691333B2 JP 2004216535 A JP2004216535 A JP 2004216535A JP 2004216535 A JP2004216535 A JP 2004216535A JP 4691333 B2 JP4691333 B2 JP 4691333B2
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- methyl
- hydrogen
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- 239000000126 substance Substances 0.000 title claims description 98
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- 150000001720 carbohydrates Chemical class 0.000 title claims description 48
- 238000005259 measurement Methods 0.000 title claims description 17
- 238000002513 implantation Methods 0.000 title claims description 16
- 238000001514 detection method Methods 0.000 claims description 43
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- 239000000463 material Substances 0.000 claims description 33
- 239000001257 hydrogen Substances 0.000 claims description 20
- 229910052739 hydrogen Inorganic materials 0.000 claims description 20
- 229920001577 copolymer Polymers 0.000 claims description 18
- 239000000758 substrate Substances 0.000 claims description 16
- 125000000217 alkyl group Chemical group 0.000 claims description 15
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 14
- -1 carboxy ester Chemical class 0.000 claims description 13
- 125000001424 substituent group Chemical group 0.000 claims description 13
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- 125000003277 amino group Chemical group 0.000 claims description 6
- 150000002431 hydrogen Chemical class 0.000 claims description 6
- 125000002252 acyl group Chemical group 0.000 claims description 5
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- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 5
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Landscapes
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Macromonomer-Based Addition Polymer (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Polyethers (AREA)
Description
本発明は、糖類の検出能に優れる蛍光モノマー化合物、蛍光センサー物質、その製造方法およびそれを用いた体内埋め込み用の糖類測定用センサー等に関する。 The present invention relates to a fluorescent monomer compound excellent in saccharide detection ability, a fluorescent sensor substance, a method for producing the same, a saccharide measuring sensor for implantation in the body using the same, and the like.
体内埋め込み型センサーは、様々な疾患においてその病状の経過観察や治療効果のモニタなどに有用であり、近年、盛んに研究されている分野の一つである。特に糖尿病治療においては、連続血糖測定による血糖コントロールが、病状の進行遅延や合併症の罹病の低減に貢献すると言われている。 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. Particularly in the treatment of diabetes, blood glucose control by continuous blood glucose measurement is said to contribute to the reduction of disease progression and morbidity of complications.
現状の糖尿病患者の多くは、血糖の自己管理のために、指等の穿刺によって血液試料を採取し、血糖計に供給して測定値を読み取ることを行っている。しかし、このような方法は患者への苦痛や簡便性のうえで問題があり、一日に数回の測定が限界で、血糖値変化の動向を頻繁に測定して把握することが難しいのが現状である。このような理由から、埋め込み型連続血糖計の有用性は高いと考えられる。 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 and grasp the blood glucose level change frequently because the measurement is limited to several times a day. Currently. For these reasons, the usefulness of the implantable continuous blood glucose meter is considered high.
一方、生体内のグルコース濃度を継続的に測定するための技術開発は古くからなされており、たとえば、可逆的にグルコースと反応して蛍光を発する物質を用いて蛍光量の変化でグルコース濃度を測定するものがある。このような蛍光物質として、発蛍光性原子団と、少なくとも1つのフェニルボロン酸部位と、少なくとも1つのアミン性窒素とを有し、アミン性窒素がフェニルボロン酸部位の近傍に配置されて該フェニルボロン酸と分子内結合する分子構造を有する発蛍光性化合物が開示されている(特許文献1)。発蛍光性原子団としては、ナフチル基やアンスリル基などがある。該化合物は、そのボロン酸部位を介して糖分子と安定なコンプレックスを形成すると蛍光を発する、というものである。 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, it has a fluorescent group, at least one phenylboronic acid moiety, and at least one amine nitrogen, and the amine nitrogen is arranged in the vicinity of the phenylboronic acid moiety. A fluorescent compound having a molecular structure in which boronic acid is bonded intramolecularly is disclosed (Patent Document 1). Examples of the fluorescent group include a naphthyl group and an anthryl group. The compound emits fluorescence when it forms a stable complex with a sugar molecule via its boronic acid moiety.
また、水性環境中での検体の濃度検出のための指示高分子として、親水性モノマーと、アントラセンホウ酸エステル誘導体などのエシキマー形成多環芳香族炭化水素とを有する化合物も開示されている(特許文献2)。該エシキマー形成多環芳香族炭化水素は水への溶解性が充分でないためメタクリルアミドなどの親水性基を導入し、水性環境中でも検体濃度を検出できるようにしたものである。 In addition, as an indicator polymer for detecting the concentration of an analyte in an aqueous environment, a compound having a hydrophilic monomer and an eschimer-forming polycyclic aromatic hydrocarbon such as an anthracene borate derivative is also disclosed (patent) Reference 2). Since the epoximer-forming polycyclic aromatic hydrocarbon is not sufficiently soluble in water, a hydrophilic group such as methacrylamide is introduced so that the analyte concentration can be detected even in an aqueous environment.
また、蛍光センサーとして、プラスチックフィルムなどの固相に直接蛍光物質を固定化する方法も開示されている(特許文献3)。特許文献3では発光性、発蛍光性または発色性の原子団にフェニルボロン酸をただ一つ付加したものを用いている。
しかしながら、上記特許文献1記載の化合物は、発蛍光性原子団としてナフチル基やアンスリル基などの嵩高くかつ疎水性の部位を有するため、水溶性の糖類との結合が容易でなく、検出感度の向上が望まれる。また、特許文献2記載の化合物は、親水性基の導入によって水溶性の糖類との結合を容易にしているが、2つの親水性基を導入しているため糖類と結合して蛍光を発する該エシキマー形成多環芳香族炭化水素が自由度を失い、糖類との結合を充分に行えない。 However, since the compound described in Patent Document 1 has a bulky and hydrophobic site such as a naphthyl group or anthryl group as a fluorescent atomic group, it is not easy to bond with a water-soluble saccharide, and detection sensitivity is low. Improvement is desired. In addition, the compound described in Patent Document 2 facilitates binding with a water-soluble saccharide by introducing a hydrophilic group. However, since two hydrophilic groups are introduced, the compound that binds to the saccharide emits fluorescence. The ethoximer-forming polycyclic aromatic hydrocarbon loses the degree of freedom and cannot fully bind to the saccharide.
一方、蛍光センサーとして使用するために蛍光物質を基材に固定すると、蛍光物質による被検出物質の検出能が固定前よりも低下する場合がある。 On the other hand, when a fluorescent substance is fixed to a substrate for use as a fluorescent sensor, the detection ability of the substance to be detected by the fluorescent substance may be lower than before fixation.
このような状況下、本発明は、グルコースなどの糖類検出能に優れる蛍光モノマー化合物、蛍光センサー物質および該蛍光センサー物質を使用した糖類測定用センサーを提供することを目的とする。 Under such circumstances, an object of the present invention is to provide a fluorescent monomer compound having excellent ability to detect saccharides such as glucose, a fluorescent sensor substance, and a saccharide measuring sensor using the fluorescent sensor substance.
本発明者は、糖類測定用蛍光モノマー化合物における糖類の結合状態を詳細に検討した結果、糖類と結合して蛍光を発する疎水性部位にポリアルキレン等からなるただ1つの親水性基を導入すると、該疎水性部位の自由度を確保しつつ糖類との結合を促進できること、更に該蛍光モノマー化合物を(メタ)アクリルアミドと共重合させると、これを基材に固定した場合であっても血液や体液などの水溶液中でも検出感度を下げることなく、糖類の測定ができることを見出し、本発明を完成させた。 As a result of examining in detail the binding state of saccharides in the fluorescent monomer compound for measuring saccharides, the present inventor introduced only one hydrophilic group consisting of polyalkylene or the like into a hydrophobic site that binds to saccharides and emits fluorescence. Bonding with saccharides can be promoted while ensuring the degree of freedom of the hydrophobic site, and further, when the fluorescent monomer compound is copolymerized with (meth) acrylamide, even when this is immobilized on a substrate, blood and body fluids The present inventors have found that sugars can be measured without lowering the detection sensitivity even in aqueous solutions such as those described above.
本発明による、糖類測定用の蛍光モノマー化合物、蛍光センサー物質、および検出層は糖類検出能に優れる。また、体液中の糖類検出能に優れるため、長期間の埋め込みに耐える蛍光センサーとなる。 The fluorescent monomer compound, fluorescent sensor substance, and detection layer for saccharide measurement according to the present invention are excellent in saccharide detection ability. Moreover, since it is excellent in the ability to detect saccharides in body fluids, it becomes a fluorescent sensor that can withstand long-term implantation.
本発明の第一は、化学式1に示す蛍光モノマー化合物である。 The first of the present invention is a fluorescent monomer compound represented by Chemical Formula 1.
また、Xは−COO−、−OCO−、−CH2NZ−、−CH2S−、−CH2O−、−NZZ’−、−NZCO−、−CONZ−、−SO2NZ−、−NZSO2−、−O−、−S−、−SS−、−NZCOO−、−OCONZ−および−CO−からなる群より選択される置換基であり、ZおよびZ’は同一または異なっていてもよく、水素または置換されていてもよいアルキル基である。ただし、Zが水素の場合にのみ−NZZ’−は−NZ−となる。アルキル基としては、メチル基、エチル基、プロピル基、ブチル基またはペンチル基などがある。Xとしては−NZCO−および−CONZ−が好ましい。 X represents —COO—, —OCO—, —CH 2 NZ—, —CH 2 S—, —CH 2 O—, —NZZ′—, —NZCO—, —CONZ—, —SO 2 NZ—, — NZSO 2 -, - O -, - S -, - SS -, - NZCOO -, - OCONZ- is a substituent selected from the group consisting of and -CO-, Z and Z 'be the same or different Often, hydrogen or an optionally substituted alkyl group. However, -NZZ'- becomes -NZ- only when Z is hydrogen. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group. X is preferably -NZCO- or -CONZ-.
また、R3は水素または置換されていてもよいアルキル基である。アルキル基としては、メチル基、エチル基、プロピル基、ブチル基またはペンチル基などがある。R3としては、水素またはメチル基が好ましい。 R 3 is hydrogen or an optionally substituted alkyl group. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group. R 3 is preferably hydrogen or a methyl group.
QおよびQ’は、水素、水酸基、置換されていてもよいアルキル基、アシル基、オキシアルキル基、ハロゲン、カルボキシル基、カルボキシエステル、カルボキシアミド、シアノ基、ニトロ基、アミノ基およびアミノアルキル基からなる群より選択される置換基である。アルキル基としては、メチル基、エチル基、プロピル基、ブチル基またはペンチル基などがあり;アシル基としては、ホルミル基、アセチル基、プロピオニル基、ブチリル基またはイソブチリル基などがあり;オキシアルキル基としては、メトキシ基、エトキシ基などがあり;ハロゲンとしては、F−、Cl−、Br−、IまたはOI−などがあり;アミノアルキル基としては、メチルアミノ基、エチルアミノ基などがある。QおよびQ’は同一または異なっていてもよく、一緒になって縮合環を形成していてもよい。QおよびQ’として、ニトロ基、シアノ基またはアシル基などを導入すると、蛍光の赤色変移または励起波長ピークと蛍光波長ピークとの間隔拡大に寄与する場合がある。本発明では、QおよびQ’として水素、アセチル基またはニトロ基が好ましい。 Q and Q ′ are hydrogen, hydroxyl group, optionally substituted alkyl group, acyl group, oxyalkyl group, halogen, carboxyl group, carboxy ester, carboxyamide, cyano group, nitro group, amino group and aminoalkyl group. A substituent selected from the group consisting of Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group; examples of the acyl group include a formyl group, an acetyl group, a propionyl group, a butyryl group, and an isobutyryl group; Includes a methoxy group, an ethoxy group, and the like; halogens include F-, Cl-, Br-, I, and OI-; and aminoalkyl groups include a methylamino group, an ethylamino group, and the like. Q and Q ′ may be the same or different and may form a fused ring together. When a nitro group, a cyano group, an acyl group, or the like is introduced as Q and Q ', it may contribute to the red transition of fluorescence or the expansion of the interval between the excitation wavelength peak and the fluorescence wavelength peak. In the present invention, hydrogen, acetyl group or nitro group is preferable as Q and Q '.
化学式1において、Yは置換されていてもよい2価の有機残基である。また、Yは該有機残基中に、下記化学式2または化学式3に示す構造を含むことが好ましく、更に、他の置換基や2価の有機残基を有していてもよい。化学式2および化学式3において、nは2〜5が好ましく、より好ましくは2〜3であり、pは1〜5が好ましく、より好ましくは2〜3であり、mは1〜200が好ましく、より好ましくは20〜100である。 In Chemical Formula 1, Y is an optionally substituted divalent organic residue. Y preferably includes a structure represented by the following chemical formula 2 or 3 in the organic residue, and may further have another substituent or a divalent organic residue. In Chemical Formula 2 and Chemical Formula 3, n is preferably 2 to 5, more preferably 2 to 3, p is preferably 1 to 5, more preferably 2 to 3, and m is preferably 1 to 200. Preferably it is 20-100.
該Yは原子数1〜500のものが好ましく、より好ましくは原子数1〜12である。
化学式2または化学式3で示す2価の有機残基は、例えばエチレングリコール、プロピレングリコールなどのアルキレングリコールまたはビニルアルコールなどを重合することで、調製することができる。
Y preferably has 1 to 500 atoms, more preferably 1 to 12 atoms.
The divalent organic residue represented by Chemical Formula 2 or Chemical Formula 3 can be prepared by polymerizing, for example, alkylene glycol such as ethylene glycol or propylene glycol, or vinyl alcohol.
なお、上記のように、本発明の蛍光モノマー化合物はアントラセン骨格を含むフェニルボロン酸誘導体であり、アントラセン骨格は発蛍光性原子団として作用することが知られている。フェニルボロン酸部位と糖とが安定な複合体を形成すると、発蛍光性原子団の介在によって蛍光を発するが、本発明の蛍光モノマー化合物は2つのフェニルボロン酸を有するため、特に糖類の検出感度に優れている。なお、化学式1においてYに結合する−NR3COCHCH2は、該蛍光モノマー化合物が血液などの体液に溶解しないように基材その他と結合させるために導入したものである。 As described above, the fluorescent monomer compound of the present invention is a phenylboronic acid derivative containing an anthracene skeleton, and the anthracene skeleton is known to act as a fluorescent atomic group. When a phenylboronic acid moiety and a saccharide form a stable complex, it fluoresces due to the presence of a fluorescent atomic group. Since the fluorescent monomer compound of the present invention has two phenylboronic acids, the saccharide detection sensitivity is particularly high. Is excellent. In addition, —NR 3 COCHCH 2 bonded to Y in Chemical Formula 1 is introduced in order to bond the fluorescent monomer compound to a substrate or the like so as not to be dissolved in a body fluid such as blood.
本発明の第二は、少なくとも以下に記載の(I)および(II)の2種の化合物を共重合してなる糖類測定用の蛍光センサー物質である。
(I):上記化学式1で示す蛍光モノマー化合物
(II):(メタ)アクリルアミド残基を含む重合性単量体
上記蛍光モノマー化合物を用いて血液に含まれる糖類を検出するには、該蛍光モノマー化合物が、血液または体液などの水溶液に溶解したり流出したりしないように固定される必要がある。しかしながら、単に蛍光モノマー化合物を基材に固定すると、該蛍光モノマー化合物と糖との接触および結合が阻害され検出感度が低下する。本発明では、該蛍光モノマー化合物と(メタ)アクリルアミド残基を含む重合性単量体を共重合させ、該蛍光モノマー化合物に親水性のポリ(メタ)アクリルアミド鎖を導入して固定化することで蛍光モノマー化合物を不溶化し、かつ該蛍光モノマー化合物と糖との親和性を確保する。
The second of the present invention is a fluorescent sensor material for measuring sugars, which is obtained by copolymerizing at least two compounds (I) and (II) described below.
(I): Fluorescent monomer compound represented by Formula 1 (II): Polymerizable monomer containing (meth) acrylamide residue In order to detect saccharides contained in blood using the fluorescent monomer compound, the fluorescent monomer It is necessary to fix the compound so that it does not dissolve or flow out in an aqueous solution such as blood or body fluid. However, when the fluorescent monomer compound is simply fixed to the substrate, contact and binding between the fluorescent monomer compound and the sugar are inhibited, and the detection sensitivity is lowered. In the present invention, the fluorescent monomer compound and a polymerizable monomer containing a (meth) acrylamide residue are copolymerized, and a hydrophilic poly (meth) acrylamide chain is introduced and immobilized on the fluorescent monomer compound. The fluorescent monomer compound is insolubilized and the affinity between the fluorescent monomer compound and sugar is ensured.
(メタ)アクリルアミド残基を含む重合性単量体としては、得られた重合体がその構造中にアクリロイル基とアミドとを有すればよく、(メタ)アクリルアミドやそれらの誘導体が含まれる。例えば、アクリルアミド、メタクリルアミド、N,N−ジメチルアクリルアミド、N−イソプロピルアクリルアミド、N−tert−ブチルアクリルアミド、N−tris−ヒドロキシメチルアクリルアミド、N−ヒドロキシメチルアクリルアミド、N−(n−ブトキシメチル)アクリルアミド、N−アクリルオイルリジンやN−アクリルオイルヘキサメチレンジアミンなどの(メタ)アクリルオイルクロライドとアミノ酸または活性アミノ基をもつ化合物との縮合体、および化学式4に示す化合物等がある。 As the polymerizable monomer containing a (meth) acrylamide residue, the obtained polymer may have an acryloyl group and an amide in its structure, and (meth) acrylamide and derivatives thereof are included. For example, acrylamide, methacrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-tert-butylacrylamide, N-tris-hydroxymethylacrylamide, N-hydroxymethylacrylamide, N- (n-butoxymethyl) acrylamide, Examples include condensates of (meth) acryloyl chlorides such as N-acryloyl lysine and N-acryloyl hexamethylene diamine with compounds having amino acids or active amino groups, and compounds represented by Chemical Formula 4.
(メタ)アクリルアミド残基を含む重合性単量体からなる重合体は親水性が高いため、蛍光モノマー化合物と結合すると、該蛍光モノマー化合物に存在するフェニルボロン酸を含む疎水性の強い発蛍光性原子団が親水性の高い構造体中に取り込まれる。これによって血液や体液に含まれる糖類を測定する場合であっても、水溶性の糖類が容易に該発蛍光性原子団に接近および結合することができる。 A polymer composed of a polymerizable monomer containing a (meth) acrylamide residue has high hydrophilicity, and therefore, when combined with a fluorescent monomer compound, it has a strong hydrophobic fluorescent property including phenylboronic acid present in the fluorescent monomer compound. Atomic groups are incorporated into highly hydrophilic structures. Thus, even when measuring saccharides contained in blood or body fluid, water-soluble saccharides can easily approach and bind to the fluorescent atomic group.
蛍光センサー物質を構成する、該蛍光モノマー化合物(I)と該アクリルアミド残基を含む重合性単量体(II)との共重合体組成モル比((I):(II))は1:10〜1:4,000であることが好ましく、より好ましくは1:50〜1:4,000であり、特に好ましくは1:100〜1:2,000である。モル比1:10よりも蛍光モノマー化合物の割合が大きくなると、該蛍光モノマー化合物の疎水性部位の嵩高さのため自由度が失われ、糖類との相互作用が低下するおそれがある。一方、モル比1:4,000よりも蛍光モノマー化合物の割合が小さければ、蛍光強度の絶対量を確保できない場合がある。 The copolymer composition molar ratio ((I) :( II)) of the fluorescent monomer compound (I) and the polymerizable monomer (II) containing the acrylamide residue constituting the fluorescent sensor substance is 1:10. It is preferably ˜1: 4,000, more preferably 1:50 to 1: 4,000, and particularly preferably 1: 100 to 1: 2,000. When the ratio of the fluorescent monomer compound is larger than the molar ratio 1:10, the degree of freedom is lost due to the bulkiness of the hydrophobic portion 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: 4,000, the absolute amount of fluorescence intensity may not be ensured.
上記二成分からなる蛍光センサー物質の、重量平均分子量は、GPCによるポリエチレンオキサイド換算で、50,000〜500,000が好ましく、より好ましくは100,000〜300,000である。 The weight average molecular weight of the fluorescent sensor material composed of the two components is preferably 50,000 to 500,000, more preferably 100,000 to 300,000 in terms of polyethylene oxide by GPC.
一方、本発明の蛍光センサー物質は、上記蛍光モノマー化合物と(メタ)アクリルアミド残基を含む重合性単量体とに加えて他の成分を併用してもよい。このような成分としては、架橋性単量体、他の架橋性成分、水中で陽イオンとなり得るカチオン性モノマー、水中で陰イオンとなり得るアニオン性モノマー、およびイオンを持たないノニオン系モノマーがある。 On the other hand, the fluorescent sensor material of the present invention may be used in combination with other components in addition to the fluorescent monomer compound and the polymerizable monomer containing a (meth) acrylamide residue. Such components include crosslinkable monomers, other crosslinkable components, cationic monomers that can be cations in water, anionic monomers that can be anions in water, and nonionic monomers that do not have ions.
架橋性単量体としては、重合性二重結合によって蛍光センサー物質中に三次元架橋構造を導入し得るものを広く含み、使用する蛍光センサー物質の置換基によっても異なるが、N,N’−メチレンビス(メタ)アクリルアミド、N,N’−(1,2−ジヒドロキシエチレン)−ビス(メタ)アクリルアミド、ジエチレングリコールジ(メタ)アクリレート、(ポリ)エチレングリコールジ(メタ)アクリレート、トリエチレングリコールジ(メタ)アクリレート、プロピレングリコールジ(メタ)アクリレート、トリメチロールプロパンジ(メタ)アクリレート、トリメチロールプロパントリ(メタ)アクリレート、ペンタエリスリトールジ(メタ)アクリレート、ペンタエリスリトールトリ(メタ)アクリレート、ペンタエリスリトールテトラ(メタ)アクリレート、(ポリ)プロピレングリコールジ(メタ)アクリレート、グリセリントリ(メタ)アクリレート、グリセリンアクリレートメタクリレート、エチレンオキサイド変性トリメチロールプロパントリ(メタ)アクリレート、ペンタエリスリトールテトラ(メタ)アクリレート、ジペンタエリスリトールヘキサ(メタ)アクリレートなどのジビニル化合物がある。本発明では、これらの2種以上を併用してもよい。 The crosslinkable monomer widely includes those capable of introducing a three-dimensional cross-linked structure into the fluorescent sensor substance by a polymerizable double bond, and varies depending on the substituent of the fluorescent sensor substance to be used, but 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 (meta) ) Acrylate, propylene glycol di (meth) acrylate, trimethylolpropane di (meth) acrylate, trimethylolpropane tri (meth) acrylate, pentaerythritol di (meth) acrylate, pentaerythritol tri (meth) acrylate, pentaerythritol te La (meth) acrylate, (poly) propylene glycol di (meth) acrylate, glycerin tri (meth) acrylate, glycerin acrylate methacrylate, ethylene oxide modified trimethylolpropane tri (meth) acrylate, pentaerythritol tetra (meth) acrylate, dipenta There are divinyl compounds such as erythritol hexa (meth) acrylate. In the present invention, two or more of these may be used in combination.
他の架橋性成分としては、2個以上の官能基を有する化合物を広く含み、使用する蛍光センサー物質の置換基によっても異なるが、トリアリルシアヌレート、トリアリルイソシアヌレート、トリアリルホスフェート、トリアリルアミン、ポリ(メタ)アリロキシアルカン、(ポリ)エチレングリコールジグリシジルエ−テル、グリセロールジグリシジルエーテル、エチレングリコール、ポリエチレングリコール、プロピレングリコール、グリセリン、ペンタエリスリトール、エチレンジアミン、ポリエチレンイミン、グリシジル(メタ)アクリレート、イソシアヌル酸トリアリル、トリメチロールプロパンジ(メタ)アリルエーテル、テトラアリロキシエタンまたはグリセロールプロポキシトリアクリレートなども挙げることが出来る。本発明では、これらの2種以上を併用してもよい。 Other crosslinkable components widely include compounds having two or more functional groups, and depending on the substituent of the fluorescent sensor material used, 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 (meth) acrylate, Mention may also be made of triallyl isocyanurate, trimethylolpropane di (meth) allyl ether, tetraallyloxyethane or glycerol propoxytriacrylate. In the present invention, two or more of these may be used in combination.
水中で陽イオンとなり得るカチオン性モノマーとしては、ジメチルアミノエチル(メタ)アクリレート、ジエチルアミノエチル(メタ)アクリレートまたは4−ビニルピリジンなどを挙げることができる。本発明では、これらの2種以上を併用してもよい。 Examples of the cationic monomer that can become a cation in water include dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, and 4-vinylpyridine. In the present invention, two or more of these may be used in combination.
水中で陰イオンとなり得るアニオン性モノマーとしては、(メタ)アクリル酸、ビニルプロピオン酸または4−ビニルベンゼンスルホン酸などを挙げることができる。本発明では、これらの2種以上を併用してもよい。 Examples of the anionic monomer that can be an anion in water include (meth) acrylic acid, vinylpropionic acid, 4-vinylbenzenesulfonic acid, and the like. In the present invention, two or more of these may be used in combination.
イオンを持たないノニオン系モノマーとしては、2−ヒドロキシエチル(メタ)アクリレート、3−メトキシプロピル(メタ)アクリレート、4−ヒドロキシブチル(メタ)アクリレート、2−メトキシエチルアクリレートまたは1,4−シクロヘキサンジメタノールモノアクリレートなどを挙げることができる。本発明では、これらの2種以上を併用してもよい。 Nonionic monomers having no ions include 2-hydroxyethyl (meth) acrylate, 3-methoxypropyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate, 2-methoxyethyl acrylate, or 1,4-cyclohexanedimethanol A monoacrylate etc. can be mentioned. In the present invention, two or more of these may be used in combination.
またこれらの架橋性単量体、他の架橋性成分、カチオン性モノマー、アニオン性モノマーおよびノニオン系モノマーは2種以上併用してもよい。これらの他の成分の配合量は、蛍光モノマー化合物と(メタ)アクリルアミド残基を含む重合性単量体との合計量の0.1〜10モル%が好ましく、より好ましくは2〜7モル%である。これら他の成分の併用によって、三次元架橋構造を形成させることができ、また、親水性の調節、反応起点の導入などを行うことができる。なお、三次元架橋構造については後述する。 These crosslinkable monomers, other crosslinkable components, cationic monomers, anionic monomers and nonionic monomers may be used in combination of two or more. The compounding amount of these other components is preferably 0.1 to 10 mol%, more preferably 2 to 7 mol% of the total amount of the fluorescent monomer compound and the polymerizable monomer containing a (meth) acrylamide residue. It is. By using these other components in combination, a three-dimensional crosslinked structure can be formed, and hydrophilicity adjustment, introduction of a reaction starting point, and the like can be performed. The three-dimensional crosslinked structure will be described later.
本発明の蛍光センサー物質として、化学式5に示される構造を有していることが好ましい。 The fluorescent sensor material of the present invention preferably has a structure represented by Chemical Formula 5.
また、iとjとのモル比(i:j)は前記(I):(II)のモル比と同様に、1:10〜1:4,000が好ましく、より好ましくは1:50〜1:4,000であり、特に好ましくは1:100〜1:2,000である。 The molar ratio (i: j) between i and j is preferably from 1:10 to 1: 4,000, more preferably from 1:50 to 1, as with the molar ratio of (I) :( II). : 4,000, particularly preferably 1: 100 to 1: 2,000.
本発明で使用する蛍光センサー物質は、該共重合体の少なくとも一部が分子間架橋を形成し、三次元架橋構造を示していてもよい。ポリ(メタ)アクリルアミド鎖に三次元架橋を形成させると上記蛍光モノマー化合物が基材に固定され、水溶液中でも蛍光モノマー化合物を溶出させることなく糖類の検出が容易にできる。なお、本発明の蛍光モノマー化合物は上記したように糖類と結合して蛍光を発する疎水性部位を有するが、該疎水性部位はYで示す二価の有機残基を介してポリ(メタ)アクリルアミド鎖に結合されるために水溶液中で糖類と結合できる自由度が確保されている。従って、三次元架橋構造を形成しても糖類の検出感度を低下させることがない。 In the fluorescent sensor material used in the present invention, at least a part of the copolymer may form an intermolecular crosslink, and may exhibit a three-dimensional crosslink structure. When a three-dimensional cross-link is formed on the poly (meth) acrylamide chain, the fluorescent monomer compound is fixed to the substrate, and saccharides can be easily detected without eluting the fluorescent monomer compound even in an aqueous solution. The fluorescent monomer compound of the present invention has a hydrophobic moiety that emits fluorescence by binding to saccharides as described above, and the hydrophobic moiety is poly (meth) acrylamide via a divalent organic residue represented by Y. Since it is bonded to the chain, a degree of freedom for bonding with saccharides in an aqueous solution is secured. Therefore, even if a three-dimensional cross-linked structure is formed, the sugar detection sensitivity is not lowered.
本発明の蛍光モノマー化合物や蛍光センサー物質の製造方法および、三次元架橋構造の形成方法には制限がないが、以下の方法で製造することができる。 Although there is no restriction | limiting in the manufacturing method of the fluorescent monomer compound and fluorescent sensor substance of this invention, and the formation method of a three-dimensional crosslinked structure, It can manufacture with the following method.
(1)蛍光モノマー化合物の製造
化学式1で示される蛍光モノマー化合物として、R1およびR2がメチル基、QおよびQ’が水素、Xが−CONH、Yが−C6H12−、R3が水素である化合物(9,10−ビス[[N−メチル−N−(オルト−ボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸−1−(6−アクリルアミド−n−ヘキシル)アミド)の製造方法の1例として、図1に示す合成スキームに従って説明する。
(1) Production of Fluorescent Monomer Compound As the fluorescent monomer compound represented by Chemical Formula 1, R 1 and R 2 are methyl groups, Q and Q ′ are hydrogen, X is —CONH, Y is —C 6 H 12 —, R 3 Of a compound in which is hydrogen (9,10-bis [[N-methyl-N- (ortho-boronobenzyl) amino] methyl] anthracene-2-carboxylic acid-1- (6-acrylamido-n-hexyl) amide) An example of the method will be described according to the synthesis scheme shown in FIG.
原料として、メチル−9,10−ジメチルアントラセン−2−カルボン酸を使用し、これにN−ブロモスクシンイミド(NBS)を作用させてメチル−9,10−ビス(ブロモメチル)アントラセン−2−カルボン酸とし、次いで、メチルアミンを添加するとブロモメチル基がアミノメチル基となる。得られたメチル−9,10−ビス(アミノメチル)アントラセン−2−カルボン酸に2−(2−ブロモメチルフェニル)−1,3−ジオキサボナリンを作用させると、メチル−9,10−ビス[[N−メチル−N−(オルトボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸が得られる。これにアルカリを作用させて脱エステル化すると、9,10−ビス[[N−メチル−N−(オルトボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸が得られる。このカルボン酸に1−アクリルアミド−6−アミノヘキサンを結合させると、上記目的物を得ることができる。 Methyl-9,10-dimethylanthracene-2-carboxylic acid is used as a raw material, and N-bromosuccinimide (NBS) is allowed to act on this to form methyl-9,10-bis (bromomethyl) anthracene-2-carboxylic acid. Then, when methylamine is added, the bromomethyl group becomes an aminomethyl group. When 2- (2-bromomethylphenyl) -1,3-dioxabonaline is allowed to act on the obtained methyl-9,10-bis (aminomethyl) anthracene-2-carboxylic acid, methyl-9,10-bis [[[ N-methyl-N- (orthoboronobenzyl) amino] methyl] anthracene-2-carboxylic acid is obtained. When this is deesterified by the action of an alkali, 9,10-bis [[N-methyl-N- (orthoboronobenzyl) amino] methyl] anthracene-2-carboxylic acid is obtained. When 1-acrylamide-6-aminohexane is bonded to this carboxylic acid, the above-mentioned target product can be obtained.
より詳細には、メチル−9,10−ジメチルアントラセン−2−カルボン酸濃度1〜20g/L、より好ましくは10〜15g/Lの溶液を調製し、これにNBSをメチル−9,10−ジメチルアントラセン−2−カルボン酸に対するモル比で2〜2.5倍、より好ましくは2.1〜2.2倍となるように配合する。なお、上記目的化合物の合成反応においては対象化合物に好適な溶媒を使用することができ、このような溶媒としてはクロロホルム、四塩化炭素、n−ヘキサン、アセトニトリル、ジメチルホルムアミド、ジメチルスルホキシドがある。これらは2種以上を併用して使用してもよい。例えば、メチル−9,10−ジメチルアントラセン−2−カルボン酸を溶解するには、クロロホルム、四塩化炭素、アセトニトリルなどを好適に使用することができる。これらは2種以上を併用して混合溶液として使用してもよい。その際の配合割合も任意に設定できる。反応温度は60〜120℃、より好ましくは80〜100℃であり、反応時間は0.5〜6時間、より好ましくは2〜4時間である。 More specifically, a solution having a methyl-9,10-dimethylanthracene-2-carboxylic acid concentration of 1 to 20 g / L, more preferably 10 to 15 g / L, is prepared, and NBS is added to methyl-9,10-dimethyl. It mix | blends so that it may become 2 to 2.5 times by mole ratio with respect to anthracene-2-carboxylic acid, More preferably, it is 2.1 to 2.2 times. In the synthesis reaction of the target compound, a solvent suitable for the target compound can be used. Examples of such a solvent include chloroform, carbon tetrachloride, n-hexane, acetonitrile, dimethylformamide, and dimethyl sulfoxide. Two or more of these may be used in combination. For example, chloroform, carbon tetrachloride, acetonitrile and the like can be suitably used to dissolve methyl-9,10-dimethylanthracene-2-carboxylic acid. Two or more of these may be used in combination as a mixed solution. The blending ratio at that time can also be set arbitrarily. The reaction temperature is 60 to 120 ° C, more preferably 80 to 100 ° C, and the reaction time is 0.5 to 6 hours, more preferably 2 to 4 hours.
次いで、溶媒で濃度1〜30g/L、より好ましくは2〜10g/Lに溶解したメチル−9,10−ビス(ブロモメチル)アントラセン−2−カルボン酸1モルに対し、2〜30モル倍、より好ましくは6〜20モル倍のメチルアミンを混合し、反応させる。反応温度は0〜60℃、より好ましくは20〜30℃で、反応時間は1〜10時間、より好ましくは2〜5時間である。 Subsequently, 2 to 30 mol times more than 1 mol of methyl-9,10-bis (bromomethyl) anthracene-2-carboxylic acid dissolved in a solvent at a concentration of 1 to 30 g / L, more preferably 2 to 10 g / L, and more Preferably 6 to 20 moles of methylamine are mixed and reacted. The reaction temperature is 0 to 60 ° C., more preferably 20 to 30 ° C., and the reaction time is 1 to 10 hours, more preferably 2 to 5 hours.
得られたメチル−9,10−ビス(アミノメチル)アントラセン−2−カルボン酸と2−(2−ブロモメチルフェニル)−1,3−ジオキサボナリンとの反応は、メチル−9,10−ビス(アミノメチル)アントラセン−2−カルボン酸に対して、2−(2−ブロモメチルフェニル)−1,3−ジオキサボナリンがモル比で2〜8倍、より好ましくは3〜5倍となるように混合する。なお、メチル−9,10−ビス(アミノメチル)アントラセン−2−カルボン酸の濃度は10〜200g/L、より好ましくは50〜100g/Lであることが好ましい。反応温度は0〜80℃、より好ましくは20〜40℃、反応時間は1〜48時間、より好ましくは2〜24時間である。 The reaction of the obtained methyl-9,10-bis (aminomethyl) anthracene-2-carboxylic acid with 2- (2-bromomethylphenyl) -1,3-dioxabonarin was carried out by methyl-9,10-bis (amino 2- (2-Bromomethylphenyl) -1,3-dioxabonaline is mixed with the methyl) anthracene-2-carboxylic acid in a molar ratio of 2 to 8 times, more preferably 3 to 5 times. The concentration of methyl-9,10-bis (aminomethyl) anthracene-2-carboxylic acid is preferably 10 to 200 g / L, more preferably 50 to 100 g / L. The reaction temperature is 0 to 80 ° C., more preferably 20 to 40 ° C., and the reaction time is 1 to 48 hours, more preferably 2 to 24 hours.
次いで、メチル−9,10−ビス[[N−メチル−N−(オルトボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸をアルカリで加水分解する。使用するアルカリとしては水酸化ナトリウム、水酸化カリウムなどいずれのアルカリ剤を使用してもよい。反応は、温度は0〜100℃、より好ましくは20〜60℃、反応時間は1〜24時間、より好ましくは2〜6時間である。 The methyl-9,10-bis [[N-methyl-N- (orthoboronobenzyl) amino] methyl] anthracene-2-carboxylic acid is then hydrolyzed with alkali. Any alkali agent such as sodium hydroxide or potassium hydroxide may be used as the alkali to be used. In the reaction, the temperature is 0 to 100 ° C., more preferably 20 to 60 ° C., and the reaction time is 1 to 24 hours, more preferably 2 to 6 hours.
次いで、9,10−ビス[[N−メチル−N−(オルトボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸1モルに対して、1−アクリルアミド−6−アミノヘキサンを1.05〜3.0モル、より好ましくは1.2〜1.4モル、および縮合剤を1.0〜3.0モル、より好ましくは1.1〜2.0モルと混合する。縮合剤としては、ジシクロヘキシルカルボジイミド、1−[3−(ジメチルアミノ)プロピル]−3−エチルカルボジイミド等を用いることができる。反応温度は0〜60℃、より好ましくは20〜30℃、反応時間は1〜24時間、より好ましくは2〜15時間である。 Subsequently, 1-acrylamido-6-aminohexane was added in an amount of 1.05 to 1 mol per mole of 9,10-bis [[N-methyl-N- (orthoboronobenzyl) amino] methyl] anthracene-2-carboxylic acid. 3.0 moles, more preferably 1.2-1.4 moles, and the condensing agent are mixed with 1.0-3.0 moles, more preferably 1.1-2.0 moles. As the condensing agent, dicyclohexylcarbodiimide, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide, or the like can be used. The reaction temperature is 0 to 60 ° C., more preferably 20 to 30 ° C., and the reaction time is 1 to 24 hours, more preferably 2 to 15 hours.
なお、原料化合物として、アントラセン骨格に化学式1で示すQ、Q’で示す置換基として上記化合物と異なる原料化合物を使用すると、溶媒、添加剤、反応温度、反応時間および分離方法等を適宜選択することでこれらに相当する化合物を製造することができる。また、カルボン酸メチルに代えてスルホニル基をアントラセン骨格に有する化合物を使用すればXとして−SO2NH−を導入することができる。また、1−アクリルアミド−6−アミノヘキサンに代えてアミノ−(C2H6O)m−アクリルアミドを作用させると、Yとして−(C2H6O)m−を導入することができる。 In addition, when a raw material compound different from the above compound is used as a raw material compound as a substituent represented by Q and Q ′ represented by Chemical Formula 1 in the anthracene skeleton, a solvent, an additive, a reaction temperature, a reaction time, a separation method, and the like are appropriately selected. Thus, compounds corresponding to these can be produced. In addition, if a compound having a sulfonyl group in the anthracene skeleton is used instead of methyl carboxylate, —SO 2 NH— can be introduced as X. Further, amino in place of 1-acrylamido-6-amino hexane - when the action of acrylamide, Y as - - (C 2 H 6 O ) m (C 2 H 6 O) m - can be introduced.
なお、9,10−ビス[[N−メチル−N−(オルトボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸は、特開平8−53467号公報の実施例3において、アンスリルジアミンに代えてアンスリルジアミン−2−カルボン酸を使用することで調製することもできる。 In addition, 9,10-bis [[N-methyl-N- (orthoboronobenzyl) amino] methyl] anthracene-2-carboxylic acid was converted to anthryldiamine in Example 3 of JP-A-8-53467. It can also be prepared by using anthryldiamine-2-carboxylic acid instead.
(2)蛍光センサー物質の製造
化学式1で示す蛍光モノマー化合物と(メタ)アクリルアミド残基を含む重合性単量体との共重合は、溶媒の下で、重合促進剤または重合開始剤を用いることができる。溶媒としては、ジメチルスルホキシド、ジメチルホルムアミド、エチレングリコール、ジエチレングリコールなどのいずれか1種以上が好ましい。該溶媒には水を含めることができ、例えばジメチルスルホキシドやジメチルホルムアミドは水との混合溶液とすると重合の進行を促進することができる。水を混合する場合には、ジメチルスルホキシドおよび/またはジメチルホルムアミドの濃度が40〜80(v/v)%のものを用いることが好ましく、より好ましくは50〜70(v/v)%である。この範囲で重合の進行を促進し、目的の蛍光センサー物質を高収率で得ることができる。なお、溶媒濃度が40(v/v)%よりも低いと、蛍光モノマー化合物が重合開始前に析出してくる場合がある。
(2) Production of fluorescent sensor substance Copolymerization of a fluorescent monomer compound represented by Chemical Formula 1 and a polymerizable monomer containing a (meth) acrylamide residue is performed using a polymerization accelerator or a polymerization initiator in a solvent. Can do. As the solvent, one or more of dimethyl sulfoxide, dimethylformamide, ethylene glycol, diethylene glycol and the like are preferable. The solvent can contain water. For example, when dimethyl sulfoxide or dimethylformamide is mixed with water, the progress of polymerization can be promoted. When water is mixed, it is preferable to use a dimethyl sulfoxide and / or dimethylformamide having a concentration of 40 to 80 (v / v)%, more preferably 50 to 70 (v / v)%. Within this range, the progress of the polymerization is promoted, and the target fluorescent sensor substance can be obtained in a high yield. When the solvent concentration is lower than 40 (v / v)%, the fluorescent monomer compound may be precipitated before the start of polymerization.
化学式1で示す蛍光モノマー化合物と(メタ)アクリルアミド残基を含む重合性単量体との共重合に際して、更に他の成分を配合することもできる。他の成分を配合する場合、その配合量は、蛍光モノマー化合物と(メタ)アクリルアミド残基を含む重合性単量体との合計量の0.1〜10モル%が好ましく、より好ましくは2〜7モル%である。他の成分を配合する場合、重合時に、重合開始剤や重合促進剤と同時に添加しておくことが好ましい。 In the copolymerization of the fluorescent monomer compound represented by Chemical Formula 1 and the polymerizable monomer containing a (meth) acrylamide residue, other components can be further blended. When the other components are blended, the blending amount is preferably 0.1 to 10 mol%, more preferably 2 to 10% of the total amount of the fluorescent monomer compound and the polymerizable monomer containing a (meth) acrylamide residue. 7 mol%. When other components are blended, it is preferably added at the same time as the polymerization initiator and the polymerization accelerator during the polymerization.
重合開始剤としては、例えば、過硫酸アンモニウム、過硫酸ナトリウム、過硫酸カリウムまたは過硫酸アンモニウムなどの過硫酸塩;過酸化水素;アゾビス−2−メチルプロピオンアミジン塩酸塩またはアゾイソブチロニトリルなどのアゾ化合物;ベンゾイルパーオキシド、ラウロイルパーオキシド、クメンハイドロパーオキシドまたは酸化ベンゾイルなどのパーオキシド等を挙げることができ、これらの1種または2種以上を用いることができる。この際、重合促進剤として亜硫酸水素ナトリウム、亜硫酸ナトリウム、モール塩、ピロ重亜硫酸ナトリウム、ホルムアルデヒドナトリウムスルホキシレートまたはアスコルビン酸などの還元剤;エチレンジアミン、エチレンジアミン四酢酸ナトリウム、グリシンまたはN,N,N’,N’−テトラメチルエチレンジアミンなどのアミン化合物;などの1種または2種以上を併用することもできる。重合温度は15〜75℃が好ましく、より好ましくは20〜60℃、重合時間は1〜20時間、より好ましくは2〜8時間である。なお、重合開始剤として過硫酸塩と重合促進剤としてN,N,N’,N’−テトラメチルエチレンジアミンとを併用する場合には、室温で重合を行うことができる点で、特に好ましい。 Examples of the polymerization initiator include persulfates such as ammonium persulfate, sodium persulfate, potassium persulfate or ammonium persulfate; hydrogen peroxide; azo compounds such as azobis-2-methylpropionamidine hydrochloride or azoisobutyronitrile. A peroxide such as benzoyl peroxide, lauroyl peroxide, cumene hydroperoxide or benzoyl oxide, and the like, and one or more of these can be used. At this time, as a polymerization accelerator, a reducing agent such as sodium hydrogen sulfite, sodium sulfite, molle salt, sodium pyrobisulfite, formaldehyde sodium sulfoxylate or ascorbic acid; ethylenediamine, ethylenediaminesodium tetraacetate, glycine or N, N, N ′ , N′-tetramethylethylenediamine and other amine compounds; and the like can be used in combination. The polymerization temperature is preferably 15 to 75 ° C, more preferably 20 to 60 ° C, and the polymerization time is 1 to 20 hours, more preferably 2 to 8 hours. In the case of using a persulfate as a polymerization initiator and N, N, N ′, N′-tetramethylethylenediamine as a polymerization accelerator in combination, it is particularly preferable because polymerization can be performed at room temperature.
一方、上記化学式5で示す化合物は、化学式1で示す蛍光モノマー化合物と(メタ)アクリルアミド残基を含む重合性単量体との共重合によらず製造することができる。化学式1で示す蛍光モノマー化合物は複数の工程で合成されるため、化合物1で示す蛍光モノマー化合物を原料とすることなくその中間産物に他の化合物を作用させても、最終的に化学式5で示す蛍光センサー物質を製造することができる。例えば、図1のスキームで示した9,10−ビス[[N−メチル−N−(オルトボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸と(メタ)アクリルアミド残基を含む重合性単量体の重合物のアミノ化物とをカップリング試薬の存在下で作用させても、化学式5で示す蛍光センサー物質を製造することができる。また別の例として、予め(メタ)アクリルアミド残基を有する重合性単量体を重合した後、重合開始剤や重合促進剤の存在下で上記蛍光モノマー化合物と共重合させても化学式5で示す蛍光センサー物質を製造することができる。 On the other hand, the compound represented by the chemical formula 5 can be produced regardless of the copolymerization of the fluorescent monomer compound represented by the chemical formula 1 and a polymerizable monomer containing a (meth) acrylamide residue. Since the fluorescent monomer compound represented by the chemical formula 1 is synthesized in a plurality of steps, even if another compound is allowed to act on the intermediate product without using the fluorescent monomer compound represented by the compound 1 as a raw material, the chemical monomer compound is finally represented by the chemical formula 5. A fluorescent sensor material can be manufactured. For example, a polymerizable monomer comprising 9,10-bis [[N-methyl-N- (orthoboronobenzyl) amino] methyl] anthracene-2-carboxylic acid and a (meth) acrylamide residue shown in the scheme of FIG. The fluorescent sensor substance represented by Chemical Formula 5 can also be produced by reacting an amination product of a polymer of a monomer in the presence of a coupling reagent. As another example, a polymerizable monomer having a (meth) acrylamide residue is previously polymerized and then copolymerized with the fluorescent monomer compound in the presence of a polymerization initiator or a polymerization accelerator, as shown in Chemical Formula 5. A fluorescent sensor material can be manufactured.
(3)三次元架橋構造の形成
本発明の蛍光センサー物質は、三次元架橋を有していてもよいのであるが、三次元架橋の導入方法も限定されない。本発明の蛍光センサー物質に架橋成分を作用させて、蛍光センサー物質と蛍光センサー物質との少なくとも一部に分子間架橋を形成させる方法がある。
(3) Formation of three-dimensional cross-linking structure The fluorescent sensor substance of the present invention may have three-dimensional cross-linking, but the method for introducing the three-dimensional cross-linking is not limited. There is a method in which a crosslinking component is allowed to act on the fluorescent sensor material of the present invention to form intermolecular crosslinks on at least a part of the fluorescent sensor material and the fluorescent sensor material.
また、前記したように、化学式1で示す蛍光モノマー化合物と(メタ)アクリルアミド残基を含む重合性単量体との共重合に際して、反応溶媒に架橋成分を添加しても三次元架橋を形成させることができる。 Further, as described above, when the fluorescent monomer compound represented by Chemical Formula 1 and the polymerizable monomer containing a (meth) acrylamide residue are copolymerized, a three-dimensional crosslinking is formed even if a crosslinking component is added to the reaction solvent. be able to.
一方、蛍光センサー物質を、体内埋め込み用の糖類測定用センサーとして使用する場合には蛍光センサー物質の流出を防止するため、基材に固定されることが一般的であるが、このような基材として、(メタ)アクリルアミド残基を含む重合性単量体またはその重合体を使用し、化学式1で示す蛍光モノマー化合物に必要に応じて架橋成分を使用し、これらを重合させ、基材への固定と三次元架橋とを同時に行うこともできる。 On the other hand, when a fluorescent sensor substance is used as a saccharide measuring sensor for implantation in the body, it is generally fixed to a base material in order to prevent the fluorescent sensor substance from flowing out. As a polymerizable monomer containing a (meth) acrylamide residue or a polymer thereof, a fluorescent monomer compound represented by Chemical Formula 1 is optionally used with a crosslinking component, and these are polymerized to form a base material. Fixing and three-dimensional crosslinking can be performed simultaneously.
このような架橋成分としては、前記蛍光センサー物質の配合し得る他の成分の項で記載した架橋性単量体、他の架橋性成分、カチオン性モノマー、アニオン性モノマーおよびノニオン系モノマーを好ましく使用することができ、架橋性単量体および他の架橋性成分をより好ましく用いることができる。本発明では、これらの2種以上を併用してもよい。 As such a cross-linking component, the cross-linkable monomer, other cross-linkable component, cationic monomer, anionic monomer and nonionic monomer described in the section of other components which can be blended with the fluorescent sensor substance are preferably used. It is possible to use a crosslinkable monomer and other crosslinkable components more preferably. In the present invention, two or more of these may be used in combination.
本発明の第三は、上記蛍光センサー物質が基材に固定化されてなる検出層である。また、本発明の第四は、上記検出層を有する体内埋め込み用の糖類測定用センサーである。 A third aspect of the present invention is a detection layer in which the fluorescent sensor substance is immobilized on a substrate. A fourth aspect of the present invention is a saccharide measurement sensor for implantation in the body having the detection layer.
体内埋め込み用の糖類測定用センサーは、蛍光センサー物質が流出しないように基材などの固定材料に、共有結合や疎水結合によって、または電気的、その他の相互作用によって固定されていることが好ましい。本発明の蛍光センサー物質を使用した糖類検出方法の概略を、図2を用いて説明する。 The saccharide measurement sensor for implantation in the body is preferably fixed to a fixing material such as a base material by a covalent bond or a hydrophobic bond, or by electrical or other interaction so that the fluorescent sensor substance does not flow out. An outline of a saccharide detection method using the fluorescent sensor material of the present invention will be described with reference to FIG.
該センサーは、検出層10を含み、検出層10には蛍光センサー物質30が基材40を介して固定されている。蛍光センサー物質30は、少なくとも黒丸で示す蛍光モノマー化合物33と白丸で示す(メタ)アクリルアミド残基を含む重合性単量体35を含む共重合体であり、水溶液中の糖類70が蛍光モノマー化合物33と相互作用すると蛍光を発する。検出層10は、光学分離層20を有していてもよい。検出層10に光源50から波長350〜420nmの光を当て、反射された蛍光量または波長の変化を検出器60によって検出すると、蛍光量に依存した糖類の濃度を知ることができる。本発明の検出層に使用する基材としては、例えばガラス、金属などの無機材料、プラスチックフィルムなどの有機材料を広く使用することができる。糖類測定用センサーに用いる検出層の基材としては、透明性に優れ、体液中でも溶解や溶出しない材料であることが好ましく、本発明では、ガラスやプラスチックフィルムの中でもポリ(メタ)アクリルアミド膜、またはポリ(メタ)アクリレート膜などを好ましく使用することができる。なお、上述したようにポリ(メタ)アクリルアミド膜を基材として使用すると、蛍光センサー物質の基材への固定と三次元架橋の形成とが同時に行える点で有利である。なお、架橋性重合体を使用した場合の架橋構造は、図2に示すように、(メタ)アクリルアミド残基を含む重合性単量体部位35と(メタ)アクリルアミド残基を含む重合性単量体部位35との間、蛍光モノマー物質部位33と(メタ)アクリルアミド残基を含む重合性単量体部位35との間、または(メタ)アクリルアミド残基を含む重合性単量体部位35と基材40との間に形成される。 The sensor includes a detection layer 10, and a fluorescent sensor material 30 is fixed to the detection layer 10 via a substrate 40. The fluorescent sensor substance 30 is a copolymer including at least a fluorescent monomer compound 33 indicated by a black circle and a polymerizable monomer 35 containing a (meth) acrylamide residue indicated by a white circle, and the saccharide 70 in the aqueous solution is a fluorescent monomer compound 33. Fluoresce when interacting with. The detection layer 10 may have an optical separation layer 20. When light having a wavelength of 350 to 420 nm is applied to the detection layer 10 from the light source 50 and a change in the reflected fluorescence amount or wavelength is detected by the detector 60, the concentration of saccharide depending on the fluorescence amount can be known. As the substrate used for the detection layer of the present invention, for example, inorganic materials such as glass and metal, and organic materials such as plastic films can be widely used. The substrate of the detection layer used in the saccharide measurement sensor is preferably a material that is excellent in transparency and does not dissolve or elute even in body fluids. In the present invention, a poly (meth) acrylamide film among glass and plastic films, or A poly (meth) acrylate film or the like can be preferably used. As described above, the use of a poly (meth) acrylamide film as a substrate is advantageous in that the fluorescent sensor substance can be fixed to the substrate and three-dimensional crosslinking can be formed simultaneously. In addition, as shown in FIG. 2, the crosslinked structure when the crosslinkable polymer is used is a polymerizable monomer portion 35 containing a (meth) acrylamide residue and a polymerizable monomer containing a (meth) acrylamide residue. Between the body part 35, the fluorescent monomer substance part 33 and the polymerizable monomer part 35 containing a (meth) acrylamide residue, or the polymerizable monomer part 35 and a group containing a (meth) acrylamide residue. It is formed between the material 40.
蛍光センサー物質を無機材料表面に固定する場合、無機材料と蛍光センサー物質とを架橋剤などを使用して化学的に結合させることもできる。このような架橋剤としては、化学式6に示すシランカップリング剤がある。 When the fluorescent sensor substance is fixed to the surface of the inorganic material, the inorganic material and the fluorescent sensor substance can be chemically bonded using a cross-linking agent or the like. As such a crosslinking agent, there is a silane coupling agent represented by Chemical Formula 6.
上記シランカップリング剤のうち、Xがビニル基、アクリル基、メタクリル基、(メタ)アクリロイル基などの重合性二重結合を有する置換基である場合には、これをガラス、金属ななどの無機材料の表面に予め塗布しておけば、これら表面でフェニルボロン酸残基とアクリルアミド残基とを含む蛍光モノマー化合物と、(メタ)アクリルアミド残基を含む重合性単量体とを共重合させつつ直接固定化することもできる。なお、シランカップリング剤で表面処理した無機材料表面に直接蛍光センサー物質を固定することもできる。 Among the silane coupling agents, when X is a substituent having a polymerizable double bond such as a vinyl group, an acrylic group, a methacryl group, or a (meth) acryloyl group, this is an inorganic material such as glass or metal. If pre-applied to the surface of the material, a fluorescent monomer compound containing a phenylboronic acid residue and an acrylamide residue and a polymerizable monomer containing a (meth) acrylamide residue are copolymerized on these surfaces. Direct immobilization is also possible. In addition, a fluorescent sensor substance can also be directly fixed on the surface of an inorganic material surface-treated with a silane coupling agent.
一方、プラスチックフィルムなどの有機材料に蛍光センサー物質を固定するには、プラスチックフィルムに反応活性基を有する置換基を導入し、蛍光センサー物質と結合させればよい。このような反応活性基の導入方法としては、例えば、特開平5−245198号公報に記載の、プラズマ、電子線、放射線などによるグリシジル(メタ)アクリレートのグラフト重合法などがある。 On the other hand, in order to fix the fluorescent sensor substance to an organic material such as a plastic film, a substituent having a reactive group may be introduced into the plastic film and bonded to the fluorescent sensor substance. As a method for introducing such a reactive group, there is, for example, a graft polymerization method of glycidyl (meth) acrylate by plasma, electron beam, radiation or the like described in JP-A-5-245198.
これらシランカップリング処理した無機材料や反応活性基を導入したプラスチックフィルムに蛍光センサー物質をより結合しやすくするには、蛍光センサー物質の合成時に予め反応活性置換基を有する単量体を共重合成分として使用してもよく、蛍光センサー物質合成後に反応活性基を導入してもよい。このような単量体としては、蛍光センサー物質の項で記載したものを使用することができる。このような反応活性基としては、アミノ基、カルボキシル基、水酸基、ハロゲン化カルボキシル基、スルホニル基、チオール基、イソシアネート基、イソチオシアネート基、またはエポキシ基等がある。なお、シランカップリング処理した無機材料や有機材料上の反応活性基と蛍光センサー物質との結合は、適当な溶媒、触媒、縮合剤の存在下、非存在下に行うことができる。 In order to make it easier to bind the fluorescent sensor substance to these silane-coupled inorganic materials and plastic films introduced with reactive active groups, monomers having reactive substituents are pre-copolymerized during the synthesis of the fluorescent sensor substances. Or a reactive group may be introduced after the synthesis of the fluorescent sensor substance. As such a monomer, those described in the section of the fluorescent sensor substance can be used. Examples of such reactive groups include amino groups, carboxyl groups, hydroxyl groups, halogenated carboxyl groups, sulfonyl groups, thiol groups, isocyanate groups, isothiocyanate groups, and epoxy groups. In addition, the coupling | bonding of the reactive active group on the inorganic material or organic material which carried out the silane coupling process, and a fluorescence sensor substance can be performed in the presence or absence of a suitable solvent, a catalyst, and a condensing agent.
本発明の体内埋め込み用の糖類測定用センサーは、少なくとも図2に示すように、蛍光センサー物質を固定した検出層、光源および蛍光検出装置を有すればよく、これらが適当なハウジング内に配置されていればよい。 The saccharide measurement sensor for implantation in the body of the present invention may have at least a detection layer, a light source and a fluorescence detection device to which a fluorescence sensor substance is fixed, as shown in FIG. 2, and these are arranged in a suitable housing. It only has to be.
本発明の検出層が、体内埋め込み用の糖類測定用センサー内に用いられる場合、図2に示すように検出層10に光学分離層20が積層されることが好ましい。光学分離層20がセンサーの外表面側に配置されると、検出層10に含まれる蛍光センサー物質と体液成分であるラジカル、酸化性物質または還元性物質などとの接触を回避することができ、これらの体液内成分による蛍光センサー物質の劣化を防止することができる。また、光学分離層20を積層すると、光源50から発した励起光の反射や散乱による検出能の低下も防止することができる。更に、糖類以外の生体物質が光源からの励起光により励起した場合であっても、光学分離層20の存在によって光源50からの励起光外部由来の光を遮蔽し、または生体内の有色物質または蛍光物質の影響を排除することもできる。 When the detection layer of the present invention is used in a saccharide measurement sensor for implantation in the body, the optical separation layer 20 is preferably laminated on the detection layer 10 as shown in FIG. When the optical separation layer 20 is disposed on the outer surface side of the sensor, it is possible to avoid contact between the fluorescent sensor substance contained in the detection layer 10 and a radical, an oxidizing substance or a reducing substance that is a body fluid component, Deterioration of the fluorescent sensor material due to these body fluid components can be prevented. Further, when the optical separation layer 20 is laminated, it is possible to prevent a decrease in detection ability due to reflection or scattering of excitation light emitted from the light source 50. Furthermore, even when biological substances other than saccharides are excited by excitation light from the light source, the presence of the optical separation layer 20 blocks light derived from the outside of the excitation light from the light source 50, or It is also possible to eliminate the influence of fluorescent substances.
このような作用を有する光学分離層20は、光学分離層基材と不透明物質とからなる。光学分離層基材としては、架橋または化学修飾されていてもよい高分子が選択され、高分子として例えばデキストラン、ポリ(メタ)アクリルアミド、ポリ(メタ)アクリレート、ポリエチレングリコール、ポリビニルアルコール、ポリアミド、ポリウレタン、これらの混合物、またはこれらの共重合体などがある。また、光学分離層基材にはビタミンE、ポリフェノール類または金属キレート類などで修飾したり、またはこれらを担持したりしてもよい。また、不透明物質としては、カーボンブラック、フラーレン、カーボンナノチューブ、または酸化鉄などを用いることができる。 The optical separation layer 20 having such an action includes an optical separation layer base material and an opaque substance. As the optical separation layer substrate, a polymer that may be cross-linked or chemically modified is selected. Examples of the polymer include dextran, poly (meth) acrylamide, poly (meth) acrylate, polyethylene glycol, polyvinyl alcohol, polyamide, and polyurethane. , A mixture thereof, or a copolymer thereof. Further, the optical separation layer substrate may be modified with vitamin E, polyphenols, metal chelates, or the like, or may be supported with these. As the opaque substance, carbon black, fullerene, carbon nanotube, iron oxide, or the like can be used.
検出層10と光学分離層20とは共有結合、イオン結合または疎水結合などにより、化学結合によって積層させることができる。例えば、光学分離層がその基材としてデキストランを、不透明物質としてカーボンブラックを使用する場合には、溶媒にデキストランを溶解させた後にカーボンブラックを添加し、超音波処理により均一化し、これにアルカリ水溶液とエチレングリコールジグリジシルエーテルを更に添加する。ついで、検出層に噴霧器を用いて上記溶液を均一に噴霧し、加熱および乾燥して検出層に光学分離層を積層する。 The detection layer 10 and the optical separation layer 20 can be laminated by chemical bonds such as covalent bonds, ionic bonds, or hydrophobic bonds. For example, when the optical separation layer uses dextran as a base material and carbon black as an opaque substance, carbon black is added after dissolving dextran in a solvent, and the mixture is homogenized by ultrasonic treatment. And ethylene glycol diglycidyl ether are further added. Next, the solution is uniformly sprayed on the detection layer using a sprayer, heated and dried, and an optical separation layer is laminated on the detection layer.
図3に本発明の体内埋め込み用の糖類測定用センサーの外観の斜視図を示す。該体内埋め込み用の糖類測定用センサーは、その内部を液密に保つハウジング110、光学分離層または検出層のみを露出させる窓部120および体外のシステムと通信するためのアンテナ部130を有する。 FIG. 3 shows a perspective view of the appearance of the saccharide measuring sensor for implantation in the body of the present invention. The in-vivo saccharide measurement sensor has a housing 110 that keeps the inside thereof liquid-tight, a window portion 120 that exposes only the optical separation layer or the detection layer, and an antenna portion 130 that communicates with a system outside the body.
図4に該体内埋め込み用の糖類測定用センサーの内部構造を示す。光学分離層20または検出層10が内部を液密に保つために窓部120を塞ぐように設けられ、励起光を発する光源50、光源50からの光を検出層10まで導く光学導波路170、検出層10からの蛍光を検出する蛍光検出装置60、蛍光検出装置60からの信号データを処理する集積回路140および内部の電源である電池150が搭載されている。なお、アンテナ部130には、アンテナ用コイル160が配設されている。なお、図3、図4は概念図であり、実施はこれに限定するものではない。必要に応じて各構成物の大きさ、形状、配置を自由に設定できる。 FIG. 4 shows the internal structure of the saccharide measuring sensor for implantation in the body. An optical separation layer 20 or a detection layer 10 is provided so as to close the window 120 in order to keep the inside liquid-tight, and a light source 50 that emits excitation light, an optical waveguide 170 that guides light from the light source 50 to the detection layer 10, A fluorescence detection device 60 that detects fluorescence from the detection layer 10, an integrated circuit 140 that processes signal data from the fluorescence detection device 60, and a battery 150 that is an internal power source are mounted. The antenna unit 130 is provided with an antenna coil 160. 3 and 4 are conceptual diagrams, and the implementation is not limited to this. The size, shape, and arrangement of each component can be freely set as necessary.
上述の、体内埋め込み用の糖類測定用センサーを用いることにより、糖尿病患者が血糖値を自己制御する際の、煩雑性または血糖値のタイムラグの発生を回避することができる。また、体内埋め込み用の糖類測定用センサーを用いることにより、糖尿病患者以外の人々が、健康管理のための血糖値測定を簡便に行うことも可能となる。 By using the above-described saccharide measurement sensor for implantation in the body, it is possible to avoid the complication or time lag of the blood sugar level when a diabetic patient self-controls the blood sugar level. In addition, by using a saccharide measurement sensor for implantation in the body, it is possible for people other than diabetics to easily perform blood glucose measurement for health management.
以下に実施例を挙げて本発明を具体的に説明するが、これらの実施例は何ら本発明を制限するものではない。 EXAMPLES The present invention will be specifically described below with reference to examples, but these examples do not limit the present invention at all.
(実施例1:9,10−ビス[[N−メチル−N−(オルト−ボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸−1−(6−アクリルアミド−n−ヘキシル)アミド(以下F−AAmと記載)の合成)
A)メチル−9,10−ビス(ブロモメチル)アントラセン−2−カルボン酸の合成
メチル−9,10−ジメチルアントラセン−2−カルボン酸360mg、N−ブロモスクシンイミド540mg、過酸化ベンゾイル5mgを、クロロホルム4mLと四塩化炭素10mLの混合物に加え、2時間加熱還流を行った。溶媒を除去後、残渣をメタノールで抽出し、目的物430mgを得た。
Example 1: 9,10-bis [[N-methyl-N- (ortho-boronobenzyl) amino] methyl] anthracene-2-carboxylic acid-1- (6-acrylamido-n-hexyl) amide (hereinafter F- Synthesis of AAm)
A) Synthesis of methyl-9,10-bis (bromomethyl) anthracene-2-carboxylic acid 360 mg of methyl-9,10-dimethylanthracene-2-carboxylic acid, 540 mg of N-bromosuccinimide, 5 mg of benzoyl peroxide and 4 mL of chloroform The mixture was added to a mixture of 10 mL of carbon tetrachloride and heated to reflux for 2 hours. After removing the solvent, the residue was extracted with methanol to obtain 430 mg of the desired product.
B)メチル−9,10−ビス(アミノメチル)アントラセン−2−カルボン酸の合成
上記A)で得たメチル−9,10−ビス(ブロモメチル)アントラセン−2−カルボン酸400mgを60mLのクロロホルムに溶解し、2Mのメチルアミンのメタノール溶液8mLを加え、室温で4時間攪拌した。溶媒除去後、メタノール/クロロホルムを溶離液とするシリカゲルカラムで精製し、目的物235mgを得た。
B) Synthesis of methyl-9,10-bis (aminomethyl) anthracene-2-carboxylic acid 400 mg of methyl-9,10-bis (bromomethyl) anthracene-2-carboxylic acid obtained in A) above was dissolved in 60 mL of chloroform. Then, 8 mL of a 2M methylamine methanol solution was added, and the mixture was stirred at room temperature for 4 hours. After removing the solvent, the residue was purified by a silica gel column using methanol / chloroform as an eluent to obtain 235 mg of the desired product.
C)9,10−ビス[[N−メチル−N−(オルト−ボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸の合成
上記B)で得たメチル−9,10−ビス(アミノメチル)アントラセン−2−カルボン酸200mg、2−(2−ブロモメチルフェニル)−1,3−ジオキサボナリン700mg、N,N−ジイソプロピルエチルアミン0.35mLを3mLのジメチルホルムアミドに溶解し、室温で16時間攪拌した。溶媒除去後、メタノール/クロロホルムを溶離液とするシリカゲルカラムで精製し、目的物のメチルエステル194mgを得た。これを5mLのメタノールに溶解し、4Nの水酸化ナトリウム1mLを加えて室温で10時間攪拌した。塩酸で中和し、ゲル濾過によって無機塩を除去し、目的物180mgを得た。生成物の融点は121℃、DMSO−d6中の1H−NMRデータは以下の通りであった。2.15ppm(d,6H,N−CH3),4.10ppm(m,4H,N−CH2−benzene),4.45ppm(m,4H,N−CH2−anthracene),7.55−8.90ppm(m,15H,aromatic)。
C) Synthesis of 9,10-bis [[N-methyl-N- (ortho-boronobenzyl) amino] methyl] anthracene-2-carboxylic acid Methyl-9,10-bis (aminomethyl) anthracene obtained in B) above 2-Carboxylic acid 200 mg, 2- (2-bromomethylphenyl) -1,3-dioxabonaline 700 mg, and N, N-diisopropylethylamine 0.35 mL were dissolved in 3 mL dimethylformamide and stirred at room temperature 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 methyl ester. This was dissolved in 5 mL of methanol, 1 mL of 4N sodium hydroxide was added, and the mixture was stirred at room temperature for 10 hours. The mixture was neutralized with hydrochloric acid and the inorganic salt was removed by gel filtration to obtain 180 mg of the desired product. The melting point of the product was 121 ° C. and 1 H-NMR data in DMSO-d 6 were as follows. 2.15 ppm (d, 6H, N—CH 3 ), 4.10 ppm (m, 4H, N—CH 2 -benzene), 4.45 ppm (m, 4H, N—CH 2 -anthracene), 7.55- 8.90 ppm (m, 15 H, aromatic).
D)F−AAmの合成
上記C)で得た9,10−ビス[[N−メチル−N−(オルト−ボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸70mg、1−アクリルアミド−6−アミノヘキサン22mg、1−[3−(ジメチルアミノ)プロピル]−3−エチルカルボジイミド50mgを50μLのN,N−ジイソプロピルエチルアミンを含むジメチルホルムアミド5mLに溶解し、60℃で18時間攪拌した。反応混合物をクロロホルム50mLに溶解し、蒸留水で3回、飽和食塩水で1回洗浄し、クロロホルム層を無水硫酸ナトリウムで乾燥後、減圧乾固し、目的物85mgを得た。
D) Synthesis of F-AAm 9,10-bis [[N-methyl-N- (ortho-boronobenzyl) amino] methyl] anthracene-2-carboxylic acid 70 mg, 1-acrylamido-6-amino obtained in C) above Hexane 22 mg and 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide 50 mg were dissolved in 5 mL of dimethylformamide containing 50 μL of N, N-diisopropylethylamine and stirred at 60 ° C. for 18 hours. The reaction mixture was dissolved in 50 mL of chloroform, washed 3 times with distilled water and once with saturated brine, and the chloroform layer was dried over anhydrous sodium sulfate and then dried under reduced pressure to obtain 85 mg of the desired product.
(実施例2:9,10−ビス[[N−メチル−N−(オルト−ボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸−(末端アクリルアミド−PEG3400)アミドの合成)
実施例1−C)で得た9,10−ビス[[N−メチル−N−(オルト−ボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸10mg、アミノ−PEG3400−アクリルアミド(ネクター社製)50mg、1−[3−(ジメチルアミノ)プロピル]−3−エチルカルボジイミド22mgを3mL100mMリン酸緩衝液(pH=6.0)に溶解し、60℃で24時間攪拌した。反応混合物をゲル濾過に供し蛍光高分子画分を採取し、目的物36mgを得た。
Example 2: Synthesis of 9,10-bis [[N-methyl-N- (ortho-boronobenzyl) amino] methyl] anthracene-2-carboxylic acid- (terminal acrylamide-PEG3400) amide
9,10-bis [[N-methyl-N- (ortho-boronobenzyl) amino] methyl] anthracene-2-carboxylic acid 10 mg obtained in Example 1-C), 50 mg of amino-PEG3400-acrylamide (manufactured by Nectar) , 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (22 mg) was dissolved in 3 mL of 100 mM phosphate buffer (pH = 6.0) and stirred at 60 ° C. for 24 hours. The reaction mixture was subjected to gel filtration, and the fluorescent polymer fraction was collected to obtain 36 mg of the desired product.
(実施例3〜9)
F−AAm濃度が10質量%であるジメチルスルホキシド(以下DMSO)溶液、アクリルアミド(以下AAmと記載)濃度が30質量%の80質量%DMSO−水混合溶液、過硫酸ナトリウム(以下SPS)濃度が3質量%の50質量%DMSO−水混合溶液、N,N,N’,N’−テトラメチルメエチレンジアミン(以下TEMED)濃度が2質量%のDMSO溶液を調製した。これら試薬溶液、DMSOおよび水を用いて最終濃度が表1の組成となるよう反応溶液を調製した。調製した該反応溶液を室温で2時間重合させF−AAmとAAmとの共重合体を得た。これらを実施例3〜9とする。得られた共重合体を、アセトンに析出・分取し、水への溶解、アセトンへの再析出を2回繰り返し精製した。精製後、得られた蛍光センサー物質を真空乾燥した。
(Examples 3 to 9)
A dimethyl sulfoxide (hereinafter DMSO) solution having an F-AAm concentration of 10% by mass, an 80% by mass DMSO-water mixed solution having an acrylamide (hereinafter referred to as AAm) concentration of 30% by mass, and a sodium persulfate (hereinafter SPS) concentration of 3 A 50% by mass DMSO-water mixed solution of 5% by mass, and a DMSO solution having a concentration of 2% by mass of N, N, N ′, N′-tetramethylmethylethylenediamine (hereinafter TEMED) were prepared. Using these reagent solutions, DMSO and water, a reaction solution was prepared so that the final concentration would be the composition shown in Table 1. The prepared reaction solution was polymerized at room temperature for 2 hours to obtain a copolymer of F-AAm and AAm. Let these be Examples 3-9. The obtained copolymer was precipitated and fractionated in acetone, and purified by repeating twice dissolution in water and reprecipitation in acetone. After purification, the resulting fluorescent sensor material was vacuum dried.
9,10−ビス[[N−メチル−N−(オルト−ボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸−(末端アクリルアミド−PEG3400)アミドが10質量%である水溶液、AAmが30質量%である水溶液、SPSが3質量%である水混合溶液、およびTEMEDが2質量%である水溶液を調製した。これら試薬溶液、DMSOおよび水を用いて最終濃度で実施例2で合成した蛍光モノマー化合物が0.6質量%、AAmが4.5質量%、SPSが0.3質量%、TEMEDが0.08質量%反応溶液を調製(仕込みモル比で1/427)し、室温で8時間重合させ共重合体を得た。精製は、アセトンに析出・分取し、水への溶解、アセトンへの再析出を10回繰り返し精製した。精製後、得られた蛍光センサー物質を真空乾燥した。
9,10-bis [[N-methyl-N- (ortho-boronobenzyl) amino] methyl] anthracene-2-carboxylic acid- (terminal acrylamide-PEG3400) amide is 10% by mass, AAm is 30% by mass A certain aqueous solution, a water mixed solution with SPS of 3% by mass, and an aqueous solution with TEMED of 2% by mass were prepared. The fluorescent monomer compound synthesized in Example 2 at a final concentration using these reagent solutions, DMSO, and water was 0.6 mass%, AAm was 4.5 mass%, SPS was 0.3 mass%, and TEMED was 0.08. A mass% reaction solution was prepared (in a charged molar ratio of 1/427) and polymerized at room temperature for 8 hours to obtain a copolymer. For purification, precipitation and fractionation were carried out in acetone, and purification was repeated 10 times by dissolving in water and reprecipitation in acetone. After purification, the resulting fluorescent sensor material was vacuum dried.
(実施例11)
実施例3〜10で得た共重合体をメタノール/リン酸緩衝液=1/2(v/v)溶液に0.05mg/mLの濃度となるよう溶解し、分光光度計を用いて265nmにおける吸光度を測定した。アクリルアミドホモポリマー(分子量150,000)についても同様に吸光度を測定し、これをBLANK値として各実施例の値から差し引いた。
予め作成した蛍光モノマー化合物の検量線から蛍光センサー物質中の蛍光モノマー化合物/AAm組成比を求めた結果を表2に示す。
(Example 11)
The copolymer obtained in Examples 3 to 10 was dissolved in a methanol / phosphate buffer solution = 1/2 (v / v) solution to a concentration of 0.05 mg / mL, and was measured at 265 nm using a spectrophotometer. Absorbance was measured. For acrylamide homopolymer (molecular weight 150,000), the absorbance was measured in the same manner, and this was subtracted from the value of each example as a BLANK value.
Table 2 shows the result of calculating the fluorescent monomer compound / AAm composition ratio in the fluorescent sensor material from the calibration curve of the fluorescent monomer compound prepared in advance.
表2から、実施例3〜9では仕込みF−AAm含量に比例して吸光度が増加しており、F−AAmが一定割合で各蛍光センサー物質成分として取り込まれていることがわかる。 From Table 2, it can be seen that in Examples 3 to 9, the absorbance increased in proportion to the charged F-AAm content, and F-AAm was incorporated as a component of each fluorescent sensor substance at a constant rate.
実施例3〜10で合成した共重合体中の蛍光モノマー化合物の濃度を揃えて蛍光強度のグルコース濃度に対する応答性を調べるため、共重合体をpH7.0のリン酸緩衝液に溶解し、265nmにおける吸光度が0.05となるように濃度調製した。グルコース濃度500mg/dLにおける各蛍光センサー物質溶液の相対蛍光強度(Ex=405nm、Em=442nm)は図5に示すように、吸光度一定、すなわち共重合体溶液中の蛍光モノマー化合物濃度一定の条件下において、実施例3(F−AAm/AAm=1/14)の蛍光センサー物質の相対蛍光比強度は、同じF−AAm/AAm共重合体系蛍光センサー物質のなかで低かった。一方、蛍光モノマー化合物の「Y」部分を長くした実施例10は非常に高い相対蛍光比強度を示した。
In order to investigate the responsiveness of the fluorescence intensity to the glucose concentration by aligning the concentration of the fluorescent monomer compound in the copolymers synthesized in Examples 3 to 10, the copolymer was dissolved in a phosphate buffer solution at pH 7.0, and 265 nm. The concentration was adjusted so that the absorbance at 0.05 was 0.05. As shown in FIG. 5, the relative fluorescence intensity (Ex = 405 nm, Em = 442 nm) of each fluorescent sensor substance solution at a glucose concentration of 500 mg / dL is a condition where the absorbance is constant, that is, the concentration of the fluorescent monomer compound in the copolymer solution is constant. The relative fluorescence specific intensity of the fluorescent sensor material of Example 3 (F-AAm / AAm = 1/14) was low among the same F-AAm / AAm copolymer fluorescent sensor materials. On the other hand, Example 10 in which the “Y” portion of the fluorescent monomer compound was lengthened showed a very high relative fluorescence specific intensity.
(実施例13)
実施例3〜9で合成した共重合体の濃度を10μg/mLと一定にする以外は実施例12と同様にして、グルコース濃度500mg/dLに対する応答性を調べた。結果を図6に示す。
(Example 13)
Responsiveness to a glucose concentration of 500 mg / dL was examined in the same manner as in Example 12 except that the concentration of the copolymer synthesized in Examples 3 to 9 was kept constant at 10 μg / mL. The results are shown in FIG.
(実施例14)
最終濃度として、AAm濃度が15質量%、N,N’−メチレンビスアクリルアミド(以下BISと記載)濃度が0.15質量%、SPS濃度が0.3質量%、TEMED濃度が0.08質量%およびF−AAm濃度が0.50質量%(仕込みモル比でF−AAm/AAm=1/300)となるよう80質量%DMSO水溶液に溶解・混合して溶液を調製した。
(Example 14)
As final concentrations, AAm concentration is 15% by mass, N, N′-methylenebisacrylamide (hereinafter referred to as BIS) concentration is 0.15% by mass, SPS concentration is 0.3% by mass, and TEMED concentration is 0.08% by mass. And it melt | dissolved and mixed in 80 mass% DMSO aqueous solution so that F-AAm density | concentration might be 0.50 mass% (F-AAm / AAm = 1/300 in a preparation molar ratio), and prepared the solution.
予め、シランカップリング剤による表面処理を行ったガラスプレートと無処理のガラスプレートを隙間を設けて設置し、隙間に上記溶液を流し込み、窒素雰囲気下室温で2時間重合させた。重合終了後ガラスプレートを純水につけて、無処理のガラスプレートのみを剥がして、ガラス基材上にF−AAm/AAmの共重合体が固定されたゲルシートを得た。得られたゲルシートを、メタノールと50mMのリン酸緩衝液(pH=7.0)とに5分間交互に浸漬を3回繰り返した後、リン酸緩衝液に10時間以上浸漬洗浄し、検出層を得た。 A glass plate that had been surface-treated with a silane coupling agent in advance and an untreated glass plate were placed with a gap between them, and the solution was poured into the gap and polymerized at room temperature in a nitrogen atmosphere for 2 hours. After completion of the polymerization, the glass plate was immersed in pure water, and only the untreated glass plate was peeled off to obtain a gel sheet in which the F-AAm / AAm copolymer was fixed on the glass substrate. The obtained gel sheet was immersed in methanol and 50 mM phosphate buffer solution (pH = 7.0) alternately for 5 minutes, and then immersed and washed in phosphate buffer solution for 10 hours or more. Obtained.
(実施例15)
実施例1−C)で合成した9,10−ビス[[N−メチル−N−(オルト−ボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸350mg、メチル−6−アミノヘキサネート200mg、1−[3−(ジメチルアミノ)プロピル]−3−エチルカルボジイミド220mgを0.2mLのN,N−ジイソプロピルエチルアミンを含むジメチルホルムアミド30mLに溶解し、室温で4時間攪拌した。反応混合物をクロロホルム100mLに溶解し、蒸留水で3回、飽和食塩水で1回洗浄し、クロロホルム層を無水硫酸ナトリウムで乾燥後、減圧乾固し、中間体であるメチルエステル360mgを得た。さらにこの中間体をメタノール10mLに溶解し、4N水酸化ナトリウム水溶液1mLを加えて室温で15時間反応させた。反応混合物をイオン交換カラムに供し、アルカリを除去し、濃縮乾固して9,10−ビス[[N−メチル−N−(オルトボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸−1−(6’−カルボン酸−n−ヘキシル)アミド310mgを得た。
(Example 15)
9,10-bis [[N-methyl-N- (ortho-boronobenzyl) amino] methyl] anthracene-2-carboxylic acid 350 mg synthesized in Example 1-C), methyl 6-aminohexanate 200 mg, 1- 220 mg of [3- (dimethylamino) propyl] -3-ethylcarbodiimide was dissolved in 30 mL of dimethylformamide containing 0.2 mL of N, N-diisopropylethylamine and stirred at room temperature for 4 hours. The reaction mixture was dissolved in 100 mL of chloroform, washed 3 times with distilled water and once with saturated brine, and the chloroform layer was dried over anhydrous sodium sulfate and then dried under reduced pressure to obtain 360 mg of an intermediate methyl ester. Further, this intermediate was dissolved in 10 mL of methanol, and 1 mL of 4N aqueous sodium hydroxide solution was added and reacted at room temperature for 15 hours. The reaction mixture is subjected to an ion exchange column to remove alkali, concentrated to dryness, and 9,10-bis [[N-methyl-N- (orthoboronobenzyl) amino] methyl] anthracene-2-carboxylic acid-1 310 mg of-(6'-carboxylic acid-n-hexyl) amide was obtained.
(実施例16)
8gの過塩素酸水溶液(有効塩素濃度5質量%)と30mLの6N水酸化ナトリウム水溶液を10cm×10cmの浅い角型ステンレス容器に取り、0℃に冷却した。10cm四方に切断したポリアクリルアミド膜を静かに浸し、0℃で2時間反応させた。反応液を除去し、40mLの蒸留水で4回、20mLのジメチルホルムアミドで2回静かに洗浄を行い活性化ポリアクリルアミド膜を得た。
(Example 16)
8 g of a perchloric acid aqueous solution (effective chlorine concentration 5 mass%) and 30 mL of 6N sodium hydroxide aqueous solution were placed in a 10 cm × 10 cm shallow square stainless steel container and cooled to 0 ° C. A polyacrylamide membrane cut to 10 cm square was gently immersed and reacted at 0 ° C. for 2 hours. The reaction solution was removed, and the membrane was gently washed four times with 40 mL distilled water and twice with 20 mL dimethylformamide to obtain an activated polyacrylamide membrane.
実施例15で合成した、蛍光モノマー化合物である9,10−ビス[[N−メチル−N−(オルトボロノベンジル)アミノ]メチル]アントラセン−2−カルボン酸−1−(6’−カルボン酸−n−ヘキシル)アミド20mg、1−[3−(ジメチルアミノ)プロピル]−3−エチルカルボジイミド12mg、1−ヒドロキシベンゾトリアゾール8mgを、ジメチルホルムアミド10mL溶解して10cm×10cmの浅い角型ステンレス容器に入れ、上記反応で作製した活性化ポリアクリルアミド膜を浸した。室温で17時間反応させた後、20mLのジメチルホルムアミドで3回、40mLの0.01N塩酸で2回、40mLの蒸留水で3回洗浄し、50mMのリン酸緩衝液(pH=7.0)で10時間以上浸漬洗浄し、前記蛍光モノマー化合物がポリアクリルアミド膜に固定された検出層を得た。 9,10-bis [[N-methyl-N- (orthoboronobenzyl) amino] methyl] anthracene-2-carboxylic acid-1- (6′-carboxylic acid), a fluorescent monomer compound synthesized in Example 15. -20 mg of n-hexyl) amide, 12 mg of 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide, and 8 mg of 1-hydroxybenzotriazole were dissolved in 10 mL of dimethylformamide in a 10 cm × 10 cm shallow square stainless steel container. Then, the activated polyacrylamide membrane prepared by the above reaction was immersed. After reacting at room temperature for 17 hours, it was washed 3 times with 20 mL of dimethylformamide, twice with 40 mL of 0.01N hydrochloric acid and 3 times with 40 mL of distilled water, and then 50 mM phosphate buffer (pH = 7.0). And washed for 10 hours or longer to obtain a detection layer in which the fluorescent monomer compound was fixed to a polyacrylamide film.
デキストラン7gを175mLの蒸留水に50℃で攪拌溶解させ、さらにカーボンブラック5gを加えた後、均一に分散するまで超音波処理を行った。そこに、50質量%水酸化ナトリウム水溶液3.5mLとエチレングリコールジグリジシルエーテル6.5gを加え、45℃で30分間攪拌を行った。更に230mLの蒸留水を加えて、この混合溶液を噴霧器に入れた。前工程で作製した検出層を平らなガラス板に固定し、上記混合溶液を均一に噴霧し、さらに45℃のオーブンで30分乾燥を行って光学分離層を積層した検出層を得た。 7 g of dextran was stirred and dissolved in 175 mL of distilled water at 50 ° C., and 5 g of carbon black was added, followed by sonication until uniformly dispersed. Thereto were added 3.5 mL of a 50% by mass aqueous sodium hydroxide solution and 6.5 g of ethylene glycol diglycidyl ether, and the mixture was stirred at 45 ° C. for 30 minutes. An additional 230 mL of distilled water was added and the mixed solution was placed in a nebulizer. The detection layer produced in the previous step was fixed on a flat glass plate, the above mixed solution was sprayed uniformly, and further dried in an oven at 45 ° C. for 30 minutes to obtain a detection layer on which an optical separation layer was laminated.
(比較例)
最終濃度としてAAmモノマー濃度が15質量%、BIS濃度が0.15質量%、SPS濃度が0.3質量%、TEMED濃度が0.08質量%および化学式7に示す9,10−ビス[N−(6−アクリルアミド)ヘキシル[−N−(オルトボロノベンジル)アミノ]メチル]アントラセン(以下、比較化合物と記載。)濃度が0.50質量%(仕込みモル比で比較化合物/AAm=1/300)となるよう80質量%DMSO水溶液に溶解・混合した。
(Comparative example)
As final concentrations, the AAm monomer concentration was 15 mass%, the BIS concentration was 0.15 mass%, the SPS concentration was 0.3 mass%, the TEMED concentration was 0.08 mass%, and 9,10-bis [N- The concentration of (6-acrylamide) hexyl [-N- (orthoboronobenzyl) amino] methyl] anthracene (hereinafter referred to as a comparative compound) is 0.50% by mass (comparative compound / AAm = 1/300 in the charged molar ratio). ) Was dissolved and mixed in an 80 mass% DMSO aqueous solution.
予め、シランカップリング剤による表面処理を行ったガラスプレートと無処理のガラスプレートとを隙間を設けて設置し、隙間に該溶液を流し込み、窒素雰囲気下室温で2時間重合させた。重合終了後、ガラスプレートを純水につけて、無処理のガラスプレートのみを剥がして、ガラス基材上に比較化合物とAAmとの共重合体であるゲルシートを得た。得られたゲルシートを、メタノールと50mMのリン酸緩衝液(pH=7.0)とに5分間交互に浸漬を3回繰り返した後、リン酸緩衝液に10時間以上浸漬洗浄し、検出層を得た。これに、実施例16と同様にして、光学分離層を積層して、検出層とした。 A glass plate that had been surface-treated with a silane coupling agent in advance and an untreated glass plate were placed with a gap between them, and the solution was poured into the gap and polymerized at room temperature in a nitrogen atmosphere for 2 hours. After completion of the polymerization, the glass plate was immersed in pure water, and only the untreated glass plate was peeled off to obtain a gel sheet which is a copolymer of the comparative compound and AAm on the glass substrate. The obtained gel sheet was immersed in methanol and 50 mM phosphate buffer solution (pH = 7.0) alternately for 5 minutes, and then immersed and washed in phosphate buffer solution for 10 hours or more. Obtained. In the same manner as in Example 16, an optical separation layer was laminated to form a detection layer.
実施例16および比較例で得られた光学分離層を積層した検出層を評価装置に固定し、リン酸緩衝液(pH=7.0)下で各濃度のグルコース応答性を蛍光強度で評価した結果を図7に示す。
The detection layer obtained by laminating the optical separation layers obtained in Example 16 and Comparative Example was fixed to an evaluation apparatus, and the glucose responsiveness of each concentration was evaluated by fluorescence intensity under a phosphate buffer solution (pH = 7.0). The results are shown in FIG.
評価装置は、検出層を固定し内部に液体を循環することのできるセルと、セルの裏面に末端が固定された光学ファイバーの束、そしてその光学ファイバーの他方末端が蛍光分光光度計へ接続された構造になっている。光学ファイバーの束の一部は励起光をセルに送るために、残りは蛍光放射光をセルから返すために用いられる構造となっている。 The evaluation device consists of a cell in which a detection layer is fixed and liquid can be circulated, a bundle of optical fibers whose ends are fixed on the back of the cell, and the other end of the optical fibers connected to a fluorescence spectrophotometer. It has a structure. A portion of the optical fiber bundle is structured to send excitation light to the cell and the rest used to return fluorescent radiation from the cell.
本発明の1つの結合部位を持つ蛍光指示薬を標識したセンサーは、2つの結合部位を持つ蛍光指示薬を標識したセンサーに比較して、グルコースに応答性が大幅に改善されていることが明らかとなった。 It has been clarified that the sensor labeled with a fluorescent indicator having one binding site of the present invention has a significantly improved response to glucose as compared with a sensor labeled with a fluorescent indicator having two binding sites. It was.
また、糖類検出能に関しても、400nmの励起で比較例では極大蛍光波長が430nmなのに対し、実施例16では450nmと長波長にシフトしており、有効であることが示された。 In addition, regarding the ability to detect saccharides, the maximum fluorescence wavelength in the comparative example was 430 nm with excitation at 400 nm, whereas in Example 16, it was shifted to 450 nm and the long wavelength, indicating that it was effective.
10…検出層、20…光学分離層、30…蛍光センサー物質、33…蛍光モノマー化合物、35…(メタ)アクリルアミド残基を含む重合性単量体、40…基材、50…光源、60…蛍光検出装置、70…糖類、110…ハウジング、120…窓部、130…アンテナ部、140…集積回路、150…電池、160…アンテナ用コイル、170…光学導波路。 DESCRIPTION OF SYMBOLS 10 ... Detection layer, 20 ... Optical separation layer, 30 ... Fluorescence sensor substance, 33 ... Fluorescence monomer compound, 35 ... Polymerizable monomer containing (meth) acrylamide residue, 40 ... Base material, 50 ... Light source, 60 ... Fluorescence detection device, 70 ... sugar, 110 ... housing, 120 ... window, 130 ... antenna, 140 ... integrated circuit, 150 ... battery, 160 ... coil for antenna, 170 ... optical waveguide.
Claims (9)
R1およびR2は同一または異なっていてもよく、アルキル基であり、
Xは−COO−、−OCO−、−CH2NZ−、−CH2S−、−CH2O−、−NZZ’−、−NZCO−、−CONZ−、−SO2NZ−、−NZSO2−、−O−、−S−、−SS−、−NZCOO−、−OCONZ−および−CO−からなる群より選択される置換基であり、ZおよびZ’は同一または異なっていてもよく、水素またはアルキル基を示し、
R3は、水素またはアルキル基を示し、
Yは−C6H12−であるか、または化学式2もしくは化学式3に示す構造を含む基であり、
QおよびQ’は同一または異なっていてもよく、水素、水酸基、アルキル基、アシル基、オキシアルキル基、ハロゲン、カルボキシル基、カルボキシエステル、カルボキシアミド、シアノ基、ニトロ基、アミノ基およびアミノアルキル基からなる群より選択される置換基である。) A fluorescent monomer compound represented by Chemical Formula 1.
R 1 and R 2 may be the same or different and are alkyl groups;
X represents —COO—, —OCO—, —CH 2 NZ—, —CH 2 S—, —CH 2 O—, —NZZ′—, —NZCO—, —CONZ—, —SO 2 NZ—, —NZSO 2. A substituent selected from the group consisting of-, -O-, -S-, -SS-, -NZCOO-, -OCONZ- and -CO-, and Z and Z 'may be the same or different; Represents hydrogen or an alkyl group,
R 3 represents hydrogen or an alkyl group,
Y is —C 6 H 12 — or a group containing a structure represented by Chemical Formula 2 or Chemical Formula 3,
Q and Q ′ may be the same or different, and hydrogen, hydroxyl group, alkyl group, acyl group, oxyalkyl group, halogen, carboxyl group, carboxy ester, carboxyamide, cyano group, nitro group, amino group and aminoalkyl group A substituent selected from the group consisting of )
(I):請求項1に記載の蛍光モノマー化合物
(II):(メタ)アクリルアミド残基を含む重合性単量体 A fluorescent sensor material for measuring sugars, which is obtained by copolymerizing at least two compounds (I) and (II) described below.
(I): the fluorescent monomer compound according to claim 1 (II): a polymerizable monomer containing a (meth) acrylamide residue
R4は、水素またはメチル基であり、
UおよびU’は同一または異なっていてもよく、水素またはアルキル基である。) The fluorescent sensor substance according to claim 2 or 3, wherein (II) is a compound represented by Chemical Formula 4.
R 4 is hydrogen or a methyl group,
U and U ′ may be the same or different and are hydrogen or an alkyl group. )
R1およびR2は同一または異なっていてもよく、アルキル基であり、
Xは−COO−、−OCO−、−CH2NZ−、−CH2S−、−CH2O−、−NZZ’−、−NZCO−、−CONZ−、−SO2NZ−、−NZSO2−、−O−、−S−、−SS−、−NZCOO−、−OCONZ−および−CO−からなる群より選択される置換基であり、ZおよびZ’は同一または異なっていてもよく、水素またはアルキル基を示し、
R3は、水素またはアルキル基を示し、
Yは−C6H12−であるか、または化学式2もしくは化学式3に示す構造を含む基であり、
QおよびQ’は同一または異なっていてもよく、水素、水酸基、アルキル基、アシル基、オキシアルキル基、ハロゲン、カルボキシル基、エステル基、アミド基、シアノ基、ニトロ基、アミノ基およびアミノアルキル基からなる群より選択される置換基であり、
R4は、水素またはメチル基であり、
UおよびU’は同一または異なっていてもよく、水素またはアルキル基であり、
iに対するjのモル比(i:j)が、1:10〜1:4,000である。) A fluorescent sensor material for measuring sugars represented by Chemical Formula 5.
R 1 and R 2 may be the same or different and are alkyl groups;
X represents —COO—, —OCO—, —CH 2 NZ—, —CH 2 S—, —CH 2 O—, —NZZ′—, —NZCO—, —CONZ—, —SO 2 NZ—, —NZSO 2. A substituent selected from the group consisting of-, -O-, -S-, -SS-, -NZCOO-, -OCONZ- and -CO-, and Z and Z 'may be the same or different; Represents hydrogen or an alkyl group,
R 3 represents hydrogen or an alkyl group,
Y is —C 6 H 12 — or a group containing a structure represented by Chemical Formula 2 or Chemical Formula 3,
Q and Q ′ may be the same or different, and hydrogen, hydroxyl group, alkyl group, acyl group, oxyalkyl group, halogen, carboxyl group, ester group, amide group, cyano group, nitro group, amino group and aminoalkyl group A substituent selected from the group consisting of
R 4 is hydrogen or a methyl group,
U and U 'may be the same or different, Ri hydrogen or an alkyl group der,
The molar ratio of j to i (i: j) is 1:10 to 1: 4,000. )
Priority Applications (8)
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JP2004216535A JP4691333B2 (en) | 2004-07-23 | 2004-07-23 | Fluorescent monomer compound for saccharide measurement, fluorescent sensor substance for saccharide measurement, and saccharide measurement sensor for implantation in the body |
AT05015807T ATE359339T1 (en) | 2004-07-23 | 2005-07-20 | SACCHARIDE SENSING FLUORESCENT MONOMER, SACCHARIDE SENSING FLUORESCENT SENSOR SUBSTANCE AND IMPLANTABLE SACCHARIDE SENSING SENSOR |
DE602005000858T DE602005000858T2 (en) | 2004-07-23 | 2005-07-20 | Saccharide-measuring fluorescent monomer, saccharide-measuring fluorescent sensor substance and implantable saccharide-measuring sensor |
EP05015807A EP1619229B1 (en) | 2004-07-23 | 2005-07-20 | Saccharide-measuring fluorescent monomer, saccharide-measuring fluorescent sensor substance, and implantable, saccharide-measuring sensor |
CN2005100851921A CN1900212B (en) | 2004-07-23 | 2005-07-22 | Saccharide-measuring fluorescent monomer, saccharide-measuring fluorescent sensor substance, and implantable, saccharide-measuring sensor |
US11/187,821 US7388110B2 (en) | 2004-07-23 | 2005-07-25 | Saccharide-measuring fluorescent monomer, saccharide-measuring fluorescent sensor substance, and implantable, saccharide-measuring sensor |
US11/890,518 US7524985B2 (en) | 2004-07-23 | 2007-08-07 | Saccharide-measuring fluorescent monomer, saccharide-measuring fluorescent sensor substance, and implantable, saccharide-measuring sensor |
US12/222,579 US7873398B2 (en) | 2004-07-23 | 2008-08-12 | Saccharide-measuring fluorescent monomer, saccharide-measuring fluorescent sensor substance, and implantable, saccharide-measuring sensor |
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WO2009009756A2 (en) * | 2007-07-11 | 2009-01-15 | Glumetrics, Inc. | Polyviologen boronic acid quenchers for use in analyte sensors |
US8334026B2 (en) * | 2009-05-29 | 2012-12-18 | Xerox Corporation | Tunable fluorescent UV curable gel inks containing fluorescent monomers for food packaging applications |
JP5288560B2 (en) * | 2009-10-29 | 2013-09-11 | 国立大学法人広島大学 | Moisture detection method |
KR101078256B1 (en) | 2010-01-13 | 2011-10-31 | 충남대학교산학협력단 | Water-soluble hyperbranched conjugated polymer for chemical sensors and biosensors, method for producing the same and fluorescent glucose sensor containing the same |
WO2014160258A1 (en) | 2013-03-14 | 2014-10-02 | Profusa, Inc. | Oxygen sensors |
US11369295B2 (en) * | 2016-07-20 | 2022-06-28 | University Of Maryland, Baltimore | Silicone hydrogel based fluorescent assay and contact lens |
WO2018119204A1 (en) | 2016-12-21 | 2018-06-28 | Profusa, Inc. | Polymerizable near-ir dyes |
JOP20190161A1 (en) * | 2016-12-27 | 2017-06-16 | Profusa Inc | Near-ir glucose sensors |
EP3813667A4 (en) * | 2018-06-27 | 2022-03-30 | Profusa, Inc. | Near-ir glucose sensors |
CN112920420B (en) * | 2021-01-29 | 2022-06-17 | 常州大学 | LCST (lower-temperature-constant temperature) adjustable aggregation-induced emission hyperbranched polymer and preparation method and application thereof |
CN114324286B (en) * | 2022-01-07 | 2022-08-02 | 中国人民解放军军事科学院军事医学研究院 | Photosensitive cross-linking agent and application thereof |
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