Syntheses of novel dipyrrylmethene-BF2 dyes and their performance as labels in two-photon excited fluoroimmunoassay
Syntheses of novel dipyrrylmethene-BF2 dyes and their performance as labels in two-photon excited fluoroimmunoassay
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DOI：
10.1023/b:jofl.0000016284.05841.46
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发表时间：
2004-03-01
影响因子：
2.7
通讯作者：
Hänninen, PE
中科院分区：
文献类型：
--
作者：
Meltola, NJ;Soini, AE;Hänninen, PE
双光子激发荧光（TPE）已被发现在显微镜成像领域的一个强大的工具，最近也在生物分析领域。最近推出的生物亲和测定技术ArcDia（TM）TPX能够从微体积中进行无分离的超灵敏免疫测定。该分析技术是基于使用微球作为固体反应载体和双光子激发荧光检测。在ArcDia（TM）TPX技术中，观察单个微粒，并通过双光子激发荧光定量微粒表面上结合的生物分子的数量。在这里，我们提出了一种新的二吡咯基亚甲基-BF 2荧光团，已被设计用于标记在ArcDia（TM）TPX测定技术的合成和使用。标签的吸收和发射波长被调谐以允许用1064 nm微芯片激光器激发。该标记含有两个羧基残基，其中一个被活化为N-羟基琥珀酰亚胺酯，以能够标记生物分子的氨基残基。另一个羧基为游离形式以增加在水溶液中的溶解度。使用ArcDia（TM）TPX分析技术在免分离免疫测定中测试这种新的荧光标记。新标签的性能相比，最亮的荧光团之一，R-藻红蛋白（RPE）。根据结果，与RPE相比，二吡咯基亚甲基-BF 2标记提供了显著更好的信号背景比，导致更高的测定灵敏度和更宽的动态范围。对水溶液的良好溶解性和高荧光量子效率表明二吡咯基亚甲基-BF 2标记也适用于其他基于荧光的应用。
Two-photon excitation of fluorescence (TPE) has been found a powerful tool in the field of microscopy imaging and recently also in the field of bioanalytics. The recently introduced bioaffinity assay technology, ArcDia(TM) TPX, enables separation-free ultra-sensitive immunoassays from microvolumes. This assay technique is based on the use of microspheres as a solid reaction carriers and two-photon excited fluorescence detection. In the ArcDia(TM) TPX-technology, the individual microparticles are observed and the number of bound biomolecules on the microparticle surface is quantified by two-photon excited fluorescence. Here we present synthesis and use of a novel dipyrrylmethene-BF2 fluorophore that has been designed to be used as label in ArcDia(TM) TPX assay technique. The absorption and emission wavelengths of the label are tuned to allow excitation with a 1064 nm microchip laser. The label contains two-carboxylic residues, one of which is activated as N-hydroxysuccinimide ester to enable labeling of amino residues of biomolecules. The other carboxylic group is in free form to increase solubility in aqueous solutions. This new fluorescent label is tested in a separation-free immunoassay using ArcDia(TM) TPX assay technique. The performance of the new label is compared to that of one of the brightest fluorophores available, R-phycoerythrin (RPE). According to the results, the dipyrrylmethene-BF2 label provides significantly better signal-to-background ratio, leading to higher assay sensitivity and broader dynamic range compared to that of RPE. Good solubility to aqueous solutions and high fluorescence quantum efficiency, suggests the dipyrrylmethene-BF2 label is applicable also in other fluorescence-based applications.