JP2005008601A - Fluorescent compound, reagent produced by using the same and analytical instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To use an LED light source as an excitation light source in the time-resolved fluorometry or fluorometry or phosphorometry with a fluorometric instrument or a phosphorometric instrument. <P>SOLUTION: The compound is expressed by R<SB>1</SB>-(-X-COCH<SB>2</SB>COC<SB>n</SB>F<SB>2n+1</SB>)<SB>m</SB>R<SB>1</SB>-X-(-COCH<SB>2</SB>COC<SB>n</SB>F<SB>2n+1</SB>)<SB>m</SB>(R<SB>1</SB>is H, an alkyl, phenyl or a group bondable to proteins, peptides, amino acids, nucleic acids or bases; X is a condensed polycyclic aromatic carbon compound which may have cyano group, nitrate group or sulfate group as a functional group; n is an integer of 1-5; and m is 1, 2 or 3). The compound is possible to have an absorption peak at ≥370 nm wavelength and has a peak sufficiently overlapping with the light wavelength emitted by a short-wavelength LED. Accordingly, the compound is excitable with an LED to provide a time-resolved fluorometry system produced by using an LED as a light source and a rare-earth element and its ligand as a fluorescent material. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００２３】
有機化合物においては二重結合が一つおきに連なった共役二重結合の構造をもつものが多く存在する。モル吸光係数は、光を吸収する度合いを示す指標であり、これが大きいほど光を強く吸収する。共役系が大きくなればなるほど、吸収ピークが長波長側にずれ、かつ、光を吸収する度合いが大きくなることを示す。
【００２４】
これらの化合物を構成する炭素原子は、ｓｐ^２ 混成をしており、その結果、分子は平面構造となり炭素の２ｐ_ｚ 原子軌道は互いに有効に重なり合って分子全体にわたり、π電子は非局在化している。
【００２５】
さらには、官能基を付加することにより、化合物の吸収ピークが移動することが知られている。図３に記載のように、ベンゼン，フェノール，ニトロフェノールと官能基が付加されるにしたがい、吸収ピークは２５５ｎｍ，２７０ｎｍ，
３１０ｎｍと長波長側に移動する。
【００２６】
紫外可視吸収は電子の運動状態の変化，遍移に関係する。光子の持つエネルギーはｈｃ／λ（ｈ：プランク定数，ｃ：光の速さ，λ：光の波長）で表されることが知られている。共役系にはπ電子が多数存在するが、このπ電子は分子の骨格を形作っているσ結合の電子より動きやすく、π電子に光子がぶつかれば電子の運動状態を容易に変えることになる。共役系が大きくなればなるほどπ電子は動きやすくなり、エネルギーの低い光子であっても遍移させることが可能となる。遍移は光子のエネルギーが電子の状態エネルギーに変換されたことを表す。光子のエネルギーが小さいことはｈｃ／λのλが大きいことになり、共役系が存在すれば長波長側に吸収ピークがでることを示す。
【００２７】
シアン基，硝酸基，硫酸基等の共役系を伸ばす官能基を縮合多環芳香族炭化水素に付加することで、吸収ピークの長波長側へのシフトがさらになされることになる。
【００２８】
（実施例２）
以下、本発明の化合物１を用いたイムノアッセイを例示する。
【００２９】
（１）抗ヒトＣＲＰ（Ｃ反応性タンパク）抗体のビオチン標識
抗ヒトＣＲＰ抗体１ｍｇをリン酸緩衝食塩水（ＰＢＳ，ｐＨ７．４）１ｍＬで溶解する。Ｓｕｌｆｏ−ＮＨＳ−ＬＣ−Ｂｉｏｔｉｎ ０．０６２ｍｇ を混合、氷浴中に２時間静置する。その後、ＰＤ−１０カラム（ファルマシア製）を用い、ＰＢＳにて抗体画分を溶出採取、未結合のＳｕｌｆｏ−ＮＨＳ−ＬＣ−Ｂｉｏｔｉｎ を除く。ビオチン標識抗体液は０．１％にアジ化ナトリウムを加え、４℃に保存する。
【００３０】
（２）ＳＡ標識体の作製
実施例１の化合物１へのクロロスルホニル基の導入
化合物０．１ミリモルあたり０．２ｍＬのクロロ硫酸（和光純薬製）を加え、室温にて７時間攪拌した後、攪拌下の純水（氷浴中）４ｍＬに反応溶液を滴下する。生成する沈殿を遠心分離し、純水にて３回洗浄する。その後、沈殿を４５時間真空乾燥する。
【００３１】
ストレプトアビジン（ＳＡ，Ｃｈｅｍｉｃｏｎ Ｉｎｔｅｒｎａｔｉｏｎａｌ製）１０^−５ミリモルを０．１Ｍ炭酸緩衝液（ｐＨ９．１）４ｍＬに溶解する。前記の化合物１０^−３ミリモルをエタノール４０μＬに溶解し、ＳＡ液中に滴下する。混合液を室温にて１時間攪拌した後、０．０５％アジ化ナトリウム加０．１ＭＮａＨＣＯ_３ 水溶液で十分に透析した。透析後、１Ｍ ＨＣｌにてｐＨを６．８に調整し全量を６ｍＬとし、０．１％にＢＳＡを加える。これを１０^−７Ｍ ＥｕＣｌ_３・６Ｈ_２Ｏ，１％ＢＳＡ，０．１％アジ化ナトリウムを含む０．０５Ｍ トリス塩酸緩衝液（ｐＨ７．８）にて３００倍に希釈し、５６℃２時間加温の後、反応に使用する。
【００３２】
（３）抗ヒトＣＲＰ抗体によるマイクロタイタープレートコーティング
抗ヒトＣＲＰ抗体の固相化されたマイクロタイタープレートを用意する。
【００３３】
０．１Ｍ炭酸緩衝液（ｐＨ９．６）で５μｇ／ｍＬに稀釈した抗ヒトＣＲＰ抗体をマイクロタイタープレートに１００μＬ分注し、４℃一夜静置コーティングを行う。その後、０．０５％Ｔｗｅｅｎ２０加生理食塩水で洗浄した後、１％ＢＳＡ，２％シュクロース加炭酸緩衝液（ｐＨ９．１）１００μＬを加え、３７℃にて静置する。１時間後、０．０５％Ｔｗｅｅｎ２０（シグマ製）加生理食塩水で洗浄し、−２０℃で保管する。
【００３４】
（４）イムノアッセイの実施
１％ＢＳＡ加生理食塩水にて、ヒトＣＲＰ標準品を１０倍段階稀釈し、その５０μＬをマイクロタイタープレート各穴に加える。３７℃，１時間振盪後、０．０５％Ｔｗｅｅｎ２０加生理食塩水で洗浄する。その後、１μｇ／Ｌに（１）のビオチン標識抗ヒトＣＲＰ抗体を１％ＢＳＡ加生理食塩水にて稀釈し、その５０μＬを各穴に分注した。３７℃，１時間振盪後、０．０５％Ｔｗｅｅｎ２０加生理食塩水で洗浄する。その後、（２）のユーロピウム標識ＳＡを１％ＢＳＡ加生理食塩水にて稀釈し、その５０μＬを各穴に分注する。
【００３５】
室温にて３０分静置後、０．０５％Ｔｗｅｅｎ２０加生理食塩水で洗浄した。ＬＥＤを光源として搭載したマイクロタイタープレートを時間分解測定装置にて発光量を計測する。
【００３６】
結果を図４に示す。化合物は標識体として、性能よくイムノアッセイにて使用できることがわかる。
【００３７】
（実施例３）
現在、青色あるいは白色光ＬＥＤは、３７０ｎｍ以上の波長のものが主である。
【００３８】
実施例１にて作成した蛍光性化合物を用いるパルス光源による時間分解測定装置の構成図を図５に示す。
【００３９】
化合物は塩化ユウロピウム液と混合され、蛍光性化合物としてサンプル容器に分注される。マイクロコンピューターにより制御されたトリガー回路によりＬＥＤの発光と消光が一定の間隔で繰り返される。時間分解測定手法により発光から少し遅れ、出力ゲートは光電子増倍管でのフォトンカウンティングを開始させマイクロコンピューターにより入力された時間間隔の蛍光計測がなされる。
【００４０】
ＬＥＤから発せられた光はレンズあるいはトロイドミラーで集光され、蛍光性化合物を含むサンプルに照射される。サンプルから発せられた蛍光はレンズあるいはトロイドミラーで集光され、光電子増倍管で計測される。
【００４１】
時間分解測定手法はユーロピウム錯体に励起光を照射した後、錯体から発光される蛍光，燐光を測定する手法であるため、励起光の単位時間当たりの点滅を多くした方が、測定感度（測定下限値）が向上する。しかし、従来の励起光光源であるキセノンランプ，レーザ等は点滅サイクルを大きくすると光量が低下する問題があった。
【００４２】
ＬＥＤは、小さな電力で発光し、また、単位時間あたりの発光消光の繰り返しサイクルを１０Ｈｚから２０００Ｈｚと大きくしても、光量の低下等の問題なく測定感度（測定下限値）を大きくすることが可能である。一方、従来の励起光光源で点滅サイクルを大きく（細かく）できない欠点を補うため照射する励起光の光量を大きくすると、サンプル容器等から散乱光が発生し、ノイズが増加するという問題も生じる。従い、ＳＮ比を確保しつつ、測定下限を向上するためには、微弱な励起光で点滅サイクルを大きくすることがより有効である。
【００４３】
ユーロピウム錯体を形成するキレートの吸収波長をＬＥＤの吸収波長に近づけた本発明の蛍光性化合物を用いることにより、初めて点滅サイクルが１０Ｈｚから２０００Ｈｚの励起光光源を用いた時間分解測定手法が可能となる。
【００４４】
（実施例４）
実施例１にて作成した蛍光性化合物を用いる連続光源による時間分解測定装置の構成図を図６に示す。
【００４５】
化合物は塩化ユウロピウム液と混合され、蛍光性化合物としてサンプル容器に分注される。マイクロコンピューターにより制御されＬＥＤが発光し、モーターによりチョッパーが回転する。トリガー回路はチョッパーからの信号を得て、時間分解測定手法により発光から少し遅れ、出力ゲートと光電子増倍管でのフォトンカウンティングをオン，オフする。
【００４６】
ＬＥＤから発せられた光はチョッパーを通過し、レンズあるいはトロイドミラーで集光され、蛍光性化合物を含むサンプルに照射される。サンプルから発せられた蛍光はレンズあるいはトロイドミラーで集光され、光電子増倍管で計測される。
【００４７】
ＬＥＤは、小さな電力で発光し、また、連続した発光をする。チョッパーの小窓よりＬＥＤ光を通過と遮断とを行う。単位時間あたりのサンプルへの励起光照射の繰り返しは、チョッパーの小窓の数と回転数により制御する。
【００４８】
パルス光である場合、発光時および消光時にはランプ本来の波長とは異なる波長の光が発生することがあり、これらが計測時のノイズとなりうる。連続光である場合、発光時および消光時の異なる波長光の発生がなくなる。
【００４９】
このことは、パルス光を発生させるための電気回路が必要でないばかりか、発光，消光による異波長光とそれらによる散乱光を発生させることなく、低ノイズでＳＮ比の高い計測を可能にする。
【００５０】
【発明の効果】
（１）本発明によれば、希土類元素とその配位子との蛍光物質は、３７０ｎｍ以上の波長域に吸収ピークを有し、これにより青色ＬＥＤあるいは紫外ＬＥＤの発光波長に重なる波長域に吸収ピークを有することが可能になる。結果として、ＬＥＤから発せられた光はエネルギーの損失を最小として試料中の蛍光物質に吸収され、蛍光が発せられる。ＬＥＤ光を光源とし、希土類元素とその配位子との蛍光物質とした時間分解蛍光測定法およびシステムが作製される。
【００５１】
（２）本発明によれば、ＬＥＤは単一あるいは狭い波長域の光、かつ、従来のキセノンランプあるいはレーザを使用するよりは長波長の光を発する。レンズ・フィルタあるいは主にプラスチックを材料とする反応容器は短波長光が照射された場合、非特異的な蛍光あるいは散乱光等のノイズをより強く発するとされている。長波長光の使用はノイズを低減させ、装置あるいは試料容器に由来するバックグラウンドを下げ、さらには装置最小検出感度を高めることができる。
【００５２】
（３）本発明によれば、蛍光測定装置，燐光測定装置はＬＥＤを光源とすることができる。ＬＥＤは小形であり、かつ、消費電力はきわめて小さい。これにより、低コスト，小形かつ簡便な蛍光分析システムを提供できる。
【図面の簡単な説明】
【図１】本発明の化合物の合成方法の一例。
【図２】本発明の化合物１の吸収／励起スペクトル。
【図３】ベンゼン，フェノール，ニトロフェノールの吸収ピーク変化。
【図４】本発明の蛍光性化合物の発光量測定結果。
【図５】本発明の測定装置の一例。
【図６】本発明の測定装置の一例。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a compound suitable as a labeling reagent for time-resolved fluorescence measurement method, delayed phosphorescence measurement method or energy transfer fluorescence detection method used in nucleic acid detection method, immunoassay method, chemiluminescence analysis method, and in particular, a conventional excitation light source. The present invention relates to a fluorescent compound having an absorption wavelength peak longer than the emission wavelength of a laser, a xenon flash lamp or the like.
[0002]
The present invention also relates to a measurement system using an LED (Light Emission Diode / Laser Diode) having an emission intensity peak longer than the emission wavelength, such as a laser or xenon flash lamp as a conventional excitation light source.
[0003]
[Prior art]
Immunoassays or nucleic acid detection methods have been measured by a number of methods including visual and radioactivity measurement, but there are many fluorescence intensity measurement methods that label analytes with fluorescent substances or fluorescent dyes because of their simplicity. It is used. The usefulness of this method is that the fluorescent compound used for the labeling body generates fluorescence or light emission in a wavelength region different from the wavelength of the excitation light, so that background noise derived from the excitation light can be reduced. Accordingly, it is desirable to select a substance that has a wavelength peak of excitation light and a wavelength peak of fluorescence or emission that is as far apart as possible and emits fluorescence or emission for a relatively long time.
[0004]
Fluorescent dyes such as rhodamine or fluorescein have a broad fluorescence wavelength distribution range and a broad fluorescence waveform can be obtained. When simultaneous measurement of multiple substances using multiple phosphors or fluorescent dyes is performed, a conversion formula is applied to know the amount of each substance, because the fluorescence wavelength ranges overlap and it is difficult to measure each substance. Therefore, it is necessary to analyze the algorithm.
[0005]
In recent years, a time-resolved fluorescence or phosphorescence measurement method has been developed as an excellent technique, taking advantage of the long fluorescence or phosphorescence quenching time emitted from complexes of rare earth elements such as europium or samarium and their ligands. . Fluorescence or phosphorescence extinction time is long, Stokes shift is large, fluorescence or phosphorescence peak waveform is sharp, fluorescence or phosphorescence signal is detected without including noise due to excitation light, and highly sensitive analysis method It is supposed to provide. In this method, excitation light is irradiated in pulses by a xenon flash lamp or a laser, and the target fluorescence or phosphorescence is measured after a time when the fluorescence of the device or other substance disappears due to the excitation light. Patent Document 1 is an invention for improvement of this ligand. 4,7-bis (chlorosulfophenyl) -1,10-phenanthroline-2,9-dicboxylic acid (BCPDA) or 4,4′-bis (1 ″) , 1 ", 1", 2 ", 2", 3 ", 3" -heptafluoro-4 ", 6" -hexane-6 "-yl) time-resolved fluorescence or phosphorescence of chlorosulfo-o-terphenyl (BHHCT). It is described that it is used for a measurement technique.
[0006]
Since these compounds have an absorption peak around 340 nm, an excitation light source having an emission peak around 340 nm such as a xenon flash lamp or a laser is used.
[0007]
Specifically, in the laser, a nitrogen gas laser that emits light having a peak at 337 nm or an Nd: YAG laser third harmonic of 355 nm light can be used.
[0008]
[Patent Document 1]
JP-A-9-241233 [0009]
[Problems to be solved by the invention]
Since these excitation light sources are large and expensive, they cause an increase in size and cost of the apparatus. On the other hand, the development progress of short wavelength LEDs has been remarkable in recent years, and blue LEDs or ultraviolet LEDs having a peak wavelength around 405 nm to 370 nm have been developed. The LED is small and low-cost, and if it can be used as an excitation light source, a small-sized and inexpensive analyzer can be provided. From the light absorption peak of a conventional ligand or complex, 340 nm. Since the energy of irradiation light is not sufficiently absorbed by the ligand or complex, it can be easily predicted that there is a problem in terms of analytical sensitivity.
[0010]
An object of the present invention is to use a time-resolved fluorescence measurement method, delayed phosphorescence measurement, which are used in nucleic acid detection methods, immunoassay methods, chemiluminescence analysis methods using an excitation light source such as an LED having a peak of emission intensity on the longer wavelength side. Or a fluorescent compound suitable for a labeling reagent for performing an energy transfer fluorescence detection method.
[0011]
Another object of the present invention is to provide a low-cost and simple measurement system using LEDs as excitation light sources.
[0012]
[Means for Solving the Problems]
The configuration of the present invention for achieving the above object is as follows.
[0013]
Formula _{R 1 - (- X-COCH} 2 COC n F 2n + 1) m
Or R ₁ —X — (— COCH ₂ COC _n F _{2n + 1} ) _m
(Wherein R ₁ is hydrogen, an alkyl group, a phenyl group, or a group capable of binding to a protein, peptide, amino acid, nucleic acid, or base, X is a condensed polycyclic aromatic carbon compound. N is 1 to 5) And m is 1, 2 or 3.
[0014]
These compounds have a light absorption peak that sufficiently overlaps the light wavelength by the short wavelength light LED. As a result, the LED can be excited to the extent that analysis can be performed, and a time-resolved fluorescence measurement system using the LED as a light source and a rare earth element and its ligand as a fluorescent substance can be achieved.
[0015]
Further, unlike a xenon flash lamp or a nitrogen gas laser, the LED does not lose its light amount even when continuously lit. For this reason, a time-resolved fluorescence measuring apparatus using a continuous light source can be produced in combination with a light blocking system such as a chopper.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be specifically described below.
[0017]
(Example 1)
A method for synthesizing the compound of the present invention using a fused polycyclic aromatic hydrocarbon as anthracene is shown in FIG.
[0018]
The absorption spectrum and excitation spectrum of Compound 1 are shown in FIG.
[0019]
The absorption spectrum was measured with a spectrophotometer U-3300 (Hitachi High Technologies), and the excitation spectrum was measured with a fluorimeter F-4010 (Hitachi High Technologies).
[0020]
FIG. 2 shows that the absorption peak and the excitation peak are in the vicinity of 415 nm.
[0021]
Table 1 shows a list of absorption peaks of the condensed polycyclic aromatic hydrocarbons. Benzene is shown as the target. It can be seen that the absorption peak is on the longer wavelength side as the number of aromatic rings increases.
[0022]
[Table 1]

[0023]
Many organic compounds have a conjugated double bond structure in which every other double bond is connected. The molar extinction coefficient is an index indicating the degree of light absorption, and the larger this is, the stronger the light is absorbed. The larger the conjugated system, the more the absorption peak shifts to the longer wavelength side, and the greater the degree of light absorption.
[0024]
Carbon atoms constituting these compounds have a sp ² hybrid, so that the molecule is 2p _z atomic orbital of carbon becomes planar structure throughout molecules effectively overlap each other, [pi electrons delocalized Yes.
[0025]
Furthermore, it is known that the absorption peak of a compound moves by adding a functional group. As shown in FIG. 3, as the functional groups are added with benzene, phenol, nitrophenol, the absorption peaks are 255 nm, 270 nm,
It moves to 310 nm and the longer wavelength side.
[0026]
UV-visible absorption is related to the change and transition of the motion state of electrons. It is known that the energy of a photon is represented by hc / λ (h: Planck constant, c: speed of light, λ: wavelength of light). There are many π electrons in the conjugated system, but these π electrons move more easily than σ-bonded electrons forming the molecular skeleton, and if a π electron hits a π electron, the movement state of the electron is easily changed. The larger the conjugated system, the easier the π electrons move, and even photons with low energy can be shifted. Translocation represents that photon energy has been converted to electron state energy. If the photon energy is small, λ of hc / λ is large, and if a conjugated system is present, an absorption peak appears on the long wavelength side.
[0027]
By adding a functional group that extends a conjugated system such as a cyan group, a nitric acid group, and a sulfuric acid group to the condensed polycyclic aromatic hydrocarbon, the absorption peak is further shifted to the long wavelength side.
[0028]
(Example 2)
Hereinafter, an immunoassay using Compound 1 of the present invention will be exemplified.
[0029]
(1) 1 mg of biotin-labeled anti-human CRP antibody of anti-human CRP (C-reactive protein) antibody is dissolved in 1 mL of phosphate buffered saline (PBS, pH 7.4). Sulfo-NHS-LC-Biotin 0.062 mg is mixed and left in an ice bath for 2 hours. Thereafter, the antibody fraction is eluted and collected with PBS using a PD-10 column (Pharmacia), and unbound Sulfo-NHS-LC-Biotin is removed. Add 0.1% sodium azide to the biotin-labeled antibody solution and store at 4 ° C.
[0030]
(2) Preparation of SA-labeled body After introducing 0.2 mL of chlorosulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) per 0.1 mmol of the compound introduced with chlorosulfonyl group into compound 1 of Example 1, the mixture was stirred at room temperature for 7 hours. The reaction solution is added dropwise to 4 mL of pure water (in an ice bath) under stirring. The resulting precipitate is centrifuged and washed three times with pure water. The precipitate is then vacuum dried for 45 hours.
[0031]
^10-5 mmol of streptavidin (SA, Chemicon International) is dissolved in 4 mL of 0.1 M carbonate buffer (pH 9.1). ^10-3 mmol of the above compound is dissolved in 40 μL of ethanol and added dropwise to the SA solution. The mixture was stirred at room temperature for 1 hour, and then dialyzed thoroughly with 0.05% sodium azide-added 0.1M NaHCO ₃ aqueous solution. After dialysis, adjust the pH to 6.8 with 1M HCl to a total volume of 6 mL, and add BSA to 0.1%. This was diluted 300-fold with 0.05 M Tris-HCl buffer (pH 7.8) containing 10 ⁻⁷ M EuCl ₃ .6H ₂ O, 1% BSA, 0.1% sodium azide, and 56 ° C. for 2 hours. Use in reaction after warming.
[0032]
(3) Microtiter plate coating with anti-human CRP antibody A microtiter plate on which an anti-human CRP antibody is immobilized is prepared.
[0033]
Dispense 100 μL of anti-human CRP antibody diluted to 5 μg / mL with 0.1 M carbonate buffer (pH 9.6) on a microtiter plate, and perform static coating at 4 ° C. overnight. Then, after washing with 0.05% Tween 20 added physiological saline, 100 μL of 1% BSA, 2% sucrose carbonated buffer (pH 9.1) is added, and the mixture is allowed to stand at 37 ° C. After 1 hour, it is washed with 0.05% Tween 20 (manufactured by Sigma) physiological saline and stored at -20 ° C.
[0034]
(4) Execution of immunoassay Dilute human CRP standard 10-fold in 1% BSA-added physiological saline, and add 50 μL to each well of a microtiter plate. After shaking at 37 ° C. for 1 hour, the cells are washed with 0.05% Tween 20-added physiological saline. Thereafter, the biotin-labeled anti-human CRP antibody (1) was diluted to 1 μg / L with 1% BSA-added physiological saline, and 50 μL thereof was dispensed into each well. After shaking at 37 ° C. for 1 hour, the cells are washed with 0.05% Tween 20-added physiological saline. Thereafter, the europium-labeled SA of (2) is diluted with 1% BSA-added physiological saline, and 50 μL thereof is dispensed into each well.
[0035]
After standing at room temperature for 30 minutes, it was washed with 0.05% Tween 20-added physiological saline. The amount of luminescence is measured with a time-resolved measuring device on a microtiter plate equipped with an LED as a light source.
[0036]
The results are shown in FIG. It can be seen that the compound can be used as a label in an immunoassay with good performance.
[0037]
Example 3
Currently, blue or white light LEDs are mainly those having a wavelength of 370 nm or more.
[0038]
FIG. 5 shows a configuration diagram of a time-resolved measurement apparatus using a pulsed light source that uses the fluorescent compound prepared in Example 1.
[0039]
The compound is mixed with a europium chloride solution and dispensed as a fluorescent compound into a sample container. The trigger circuit controlled by the microcomputer repeats light emission and extinction of the LED at regular intervals. With the time-resolved measurement method, the output gate is slightly delayed from the light emission, and the output gate starts photon counting in the photomultiplier tube, and the fluorescence is measured at the time interval input by the microcomputer.
[0040]
The light emitted from the LED is collected by a lens or a toroid mirror and irradiated on a sample containing a fluorescent compound. The fluorescence emitted from the sample is collected by a lens or a toroid mirror and measured by a photomultiplier tube.
[0041]
The time-resolved measurement method is a method that measures the fluorescence and phosphorescence emitted from the complex after irradiating the europium complex with the excitation light. Value). However, conventional xenon lamps, lasers, and the like, which are excitation light sources, have a problem that the amount of light decreases when the blink cycle is increased.
[0042]
The LED emits light with low power, and even if the repetition cycle of light emission quenching per unit time is increased from 10 Hz to 2000 Hz, the measurement sensitivity (measurement lower limit value) can be increased without problems such as a decrease in the amount of light. It is. On the other hand, if the amount of excitation light to be irradiated is increased in order to compensate for the drawback that the blinking cycle cannot be increased (finely) with the conventional excitation light source, scattered light is generated from the sample container or the like, and noise increases. Accordingly, in order to improve the lower limit of measurement while ensuring the SN ratio, it is more effective to increase the blinking cycle with weak excitation light.
[0043]
By using the fluorescent compound of the present invention in which the absorption wavelength of the chelate forming the europium complex is close to the absorption wavelength of the LED, a time-resolved measurement method using an excitation light source with a blinking cycle of 10 Hz to 2000 Hz becomes possible for the first time. .
[0044]
(Example 4)
FIG. 6 shows a configuration diagram of a time-resolved measurement apparatus using a continuous light source that uses the fluorescent compound prepared in Example 1.
[0045]
The compound is mixed with a europium chloride solution and dispensed as a fluorescent compound into a sample container. The LED is controlled by a microcomputer and the chopper is rotated by a motor. The trigger circuit obtains the signal from the chopper, delays it slightly by the time-resolved measurement method, and turns on / off photon counting at the output gate and the photomultiplier tube.
[0046]
The light emitted from the LED passes through the chopper, is collected by a lens or a toroid mirror, and is irradiated to the sample containing the fluorescent compound. The fluorescence emitted from the sample is collected by a lens or a toroid mirror and measured by a photomultiplier tube.
[0047]
The LED emits light with a small electric power and emits light continuously. The LED light is passed and blocked from the chopper's small window. The repetition of excitation light irradiation on the sample per unit time is controlled by the number of chopper windows and the number of rotations.
[0048]
In the case of pulsed light, light having a wavelength different from the original wavelength of the lamp may be generated during light emission and extinction, and these may be noise during measurement. In the case of continuous light, generation of light having different wavelengths during light emission and extinction is eliminated.
[0049]
This not only requires an electric circuit for generating pulsed light, but also enables measurement with low noise and high S / N ratio without generating different wavelength light due to light emission and quenching and scattered light due to them.
[0050]
【The invention's effect】
(1) According to the present invention, the fluorescent material of the rare earth element and its ligand has an absorption peak in the wavelength region of 370 nm or more, and thereby absorbs in the wavelength region overlapping the emission wavelength of the blue LED or ultraviolet LED. It becomes possible to have a peak. As a result, the light emitted from the LED is absorbed by the fluorescent material in the sample with a minimum loss of energy, and fluorescence is emitted. A time-resolved fluorescence measurement method and system using LED light as a light source and a fluorescent material of a rare earth element and its ligand are produced.
[0051]
(2) According to the present invention, the LED emits light of a single or narrow wavelength range and light of a longer wavelength than using a conventional xenon lamp or laser. It is said that a reaction vessel made of a lens / filter or mainly a plastic material emits noise such as non-specific fluorescence or scattered light more strongly when irradiated with short-wavelength light. The use of long-wavelength light can reduce noise, lower the background originating from the device or sample container, and further increase the device minimum detection sensitivity.
[0052]
(3) According to the present invention, the fluorescence measuring device and the phosphorescence measuring device can use an LED as a light source. LEDs are small and consume very little power. Thereby, a low-cost, small and simple fluorescence analysis system can be provided.
[Brief description of the drawings]
FIG. 1 shows an example of a method for synthesizing a compound of the present invention.
FIG. 2: Absorption / excitation spectrum of Compound 1 of the present invention.
FIG. 3 shows changes in absorption peaks of benzene, phenol and nitrophenol.
FIG. 4 shows the result of measuring the amount of luminescence of the fluorescent compound of the present invention.
FIG. 5 shows an example of a measuring apparatus according to the present invention.
FIG. 6 shows an example of a measuring apparatus according to the present invention.

Claims (13)

Formula _{R 1 - (- X-COCH} 2 COC n F 2n + 1) m
(Wherein R ₁ is hydrogen, an alkyl group, a phenyl group, or a group capable of binding to a protein, peptide, amino acid, nucleic acid, or base, X is a condensed polycyclic aromatic carbon compound. N is 1 to 5) And m is 1, 2 or 3. Formula _{R 1 -X - (- COCH 2} COC n F 2n + 1) m
(Wherein R ₁ is hydrogen, an alkyl group, a phenyl group, or a group capable of binding to a protein, peptide, amino acid, nucleic acid, or base, X is a condensed polycyclic aromatic carbon compound. N is 1 to 5) And m is 1, 2 or 3. The fluorescent compound according to claim 1 or 2,
X is a fluorescent compound having a cyan group, a nitrate group, and a sulfate group as functional groups.

(In the formula, R ₁ is hydrogen, an alkyl group, a phenyl group, or a group capable of binding to a protein, peptide, amino acid, nucleic acid or base, and cyan at one or a plurality of positions represented by Y ₁ to Y _6. A functional group having a group, a nitric acid group and a sulfuric acid group, n is an integer from 1 to 5, and m is 1, 2 or 3.)

(In the formula, R ₁ is hydrogen, an alkyl group, a phenyl group, or a group capable of binding to a protein, peptide, amino acid, nucleic acid or base, and cyan at one or a plurality of positions represented by Y ₁ to Y _8. A functional group having a group, a nitric acid group and a sulfuric acid group, n is an integer from 1 to 5, and m is 1, 2 or 3.)

(In the formula, R ₁ is hydrogen, an alkyl group, a phenyl group, or a group capable of binding to a protein, peptide, amino acid, nucleic acid or base, and cyan at one or a plurality of positions represented by Y ₁ to Y _8. A functional group having a group, a nitric acid group and a sulfuric acid group, n is an integer from 1 to 5, and m is 1, 2 or 3.)

(In the formula, R ₁ is hydrogen, an alkyl group, a phenyl group, or a group capable of binding to a protein, peptide, amino acid, nucleic acid, or base, and cyan at one or a plurality of positions represented by Y ₁ to Y _10. A functional group having a group, a nitric acid group and a sulfuric acid group, n is an integer from 1 to 5, and m is 1, 2 or 3.)

(In the formula, R ₁ is hydrogen, an alkyl group, a phenyl group, or a group capable of binding to a protein, peptide, amino acid, nucleic acid or base, and cyan at one or a plurality of positions represented by Y ₁ to Y _8. A functional group having a group, a nitric acid group and a sulfuric acid group, n is an integer from 1 to 5, and m is 1, 2 or 3.)

(In the formula, R ₁ is hydrogen, an alkyl group, a phenyl group, or a group capable of binding to a protein, peptide, amino acid, nucleic acid, or base, and cyan at one or a plurality of positions represented by Y ₁ to Y _10. A functional group having a group, a nitric acid group and a sulfuric acid group, n is an integer from 1 to 5, and m is 1, 2 or 3.) A complex formed by the fluorescent compound according to claim 1 or 2 and a rare earth element ion, and having a maximum absorption wavelength at 370 nm or more. The labeling reagent containing the fluorescent compound or complex in any one of Claims 1-10. A light emitting diode;
A light emission control circuit for emitting the light emitting diode at a blinking cycle of 10 Hz to 1000 Hz;
An optical system for guiding light emitted from the light emitting diode to a holding means for holding a sample;
Detection means for detecting luminescence from the sample;
Analytical device equipped with. A light emitting diode;
A light emission control circuit for continuously emitting light from the light emitting diode;
A chopper that intermittently emits light from the light emitting diode with a flashing cycle of 10 Hz to 2000 Hz;
An optical system for guiding light emitted from the light emitting diode to a holding means for holding a sample;
Detection means for detecting luminescence from the sample;
Analytical device equipped with.

Images