A Ratiometric Fluorescent Probe Based on FRET for Imaging Hg2+ Ions in Living Cells
A Ratiometric Fluorescent Probe Based on FRET for Imaging Hg2+ Ions in Living Cells
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基于 FRET 的比率荧光探针用于活细胞中 Hg2 离子成像
DOI：
10.1002/anie.200803246
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Qian, Xuhong
中科院分区：
文献类型：
--
作者：
Zhang, Xiaolin;Xiao, Yi;Qian, Xuhong
汞离子很容易穿过生物膜，对中枢神经和内分泌系统造成严重损害。[1]因此，活细胞中Hg 2+离子的成像对于阐明其生物学效应至关重要。荧光光谱法由于其简单和灵敏而成为检测和成像痕量样品的有力工具。[2]因此，荧光Hg 2+探针的开发，[3]特别是那些在活细胞中具有实际应用的探针，[4]引起了人们的广泛关注。大多数报道的Hg 2+离子在活细胞中的荧光感测的实例通过增强荧光信号来起作用。然而，由于荧光强度的变化是唯一的检测信号，诸如仪器效率、环境条件和探针浓度的因素可能干扰信号输出。[5]比率式传感器可以通过两个发射带的自校准来消除大多数或所有的模糊性。[6]比率探针可以设计成遵循两种机制起作用：分子内电荷转移（ICT）和荧光共振能量转移（FRET）。ICT探针已被频繁报道，并且一些探针在生理条件下工作良好。可能影响ICT探针准确性的两个方面是：1）靶离子的结合促进或抑制ICT相互作用，这导致传感器吸收最大值的显著偏移;但是如果使用多个激发波长来匹配不同的激发最大值，则它们的效率差异可能是不准确性的潜在来源。2)ICT荧光团经常观察到相对较宽的荧光光谱;在大量情况下，结合靶离子之前和之后的宽荧光光谱具有高度重叠（或在极端情况下，具有高强度的宽光谱完全覆盖具有较低强度的宽光谱），这使得难以准确地确定两个荧光峰的比率。理论上，上述问题可以通过使用基于FRET的传感器来避免，对于该基于FRET的传感器，供体荧光团的单一激发波长导致受体在较长波长下的发射。[7]在这里，我们提出了BODIPY-罗丹明（BODIPY=硼-dipyrromethene）FRET“关-开”系统3作为比率和细胞内Hg 2+传感器。选择了一种无色罗丹明衍生物作为汞离子的敏感性和选择性化学传感器。这是受到Tae及其同事以及其他研究小组的启发，[8]他们使用这些具有非共轭结构的隐色衍生物作为荧光和显色传感器。Hg 2+诱导的高效开环反应产生长波长罗丹明荧光团，可作为能量受体。选择BODIPY [9]作为能量供体是因为其强烈的荧光对环境因素不敏感，并且其荧光光谱与罗丹明的吸收光谱匹配良好。供体和受体之间的连接的选择同样重要;刚性和共轭的苯基-乙炔基-苯基间隔基，其不仅促进了通过键的能量转移过程[7a]，而且大大简化了相对大分子的合成，被确定为理想的桥。传感器3和开环产物4都被有效地合成（方案1）并被很好地表征。Hg ~（2+）诱导的过程可使体系的最大发射波长由BODIPY的特征峰514 nm变为罗丹明的特征峰589 nm。这种波长移动允许在乙醇/水溶液中的汞离子的比率检测，并...
Mercury ions can easily pass through biological membranes and cause serious damage to the central nervous and endocrine systems.[1] Therefore, imaging of Hg2+ ions in living cells is crucial for the elucidation of their biological effects. Fluorescence spectroscopy has become a powerful tool for sensing and imaging trace amounts of samples because of its simplicity and sensitivity.[2] Thus, the development of fluorescent Hg2+ probes,[3] particularly those that have practical application in living cells,[4] has attracted much attention. Most reported examples of fluorescent sensing of Hg2+ ions in living cells function by the enhancement of fluorescence signals. However, as the change in fluorescence intensity is the only detection signal, factors such as instrumental efficiency, environmental conditions, and the probe concentration can interfere with the signal output.[5] Ratiometric sensors can eliminate most or all ambiguities by selfcalibration of two emission bands.[6] Ratiometric probes can be designed to function following two mechanisms: intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET). ICT probes have been frequently reported and some work well under physiological conditions. Two aspects which potentially influence the accuracy of ICT probes are: 1) Binding of the target ions promotes or inhibits ICT interactions, which results in remarkable shifts of the sensors absorption maxima; but if multiple excitation wavelengths are used to match the different excitation maxima, their difference in efficiency may be a potential origin of inaccuracy. 2) Relatively broad fluorescence spectra are often observed for ICT fluorophores; in a significant number of cases the broad fluorescence spectra before and after binding target ions have a high degree of overlap (or in an extreme case, a broad spectrum with high intensity completely covers one with lower intensity), which makes it difficult to accurately determine the ratio of the two fluorescence peaks. Theoretically, the above problems can be avoided by using a FRET-based sensor for which the single excitation wavelength of a donor fluorophore results in emission of the acceptor at a longer wavelength.[7] Herein we present a BODIPY-rhodamine (BODIPY= boron–dipyrromethene) FRET “off–on” system 3 as a ratiometric and intracellular Hg2+ sensor. A leuco-rhodamine derivative was chosen as a sensitive and selective chemosensor for Hg2+ ions. This was inspired by Tae and co-workers as well as other research groups,[8], who used these leuco derivatives with unconjugated structures as fluorogenic and chromogenic sensors. A highly efficient ring-opening reaction induced by Hg2+ generates the long-wavelength rhodamine fluorophore which can act as the energy acceptor. BODIPY [9] was chosen as the energy donor because its intense fluorescence is insensitive to environmental factors and its fluorescence spectrum matches well with the absorption spectrum of rhodamine. The choice of the connection between the donor and acceptor was equally important; a rigid and conjugated phenyl–ethynyl–phenyl spacer, which not only facilitates the through-bond energy transfer process [7a] but also greatly simplifies the synthesis of a relatively large molecule, was identified as an ideal bridge.Both sensor 3 and ring-opened product 4 were efficiently synthesized (Scheme1) and well characterized. An Hg2+-induced process can change the emission maximum of the system from 514 nm (the characteristic peak of BODIPY) to 589 nm (the characteristic peak of rhodamine). This wavelength shift allows the ratiometric detection of Hg2+ ions both in ethanol/water solution and in …