A Quantum-Dot-Based Molecular Ruler for Multiplexed Optical Analysis
A Quantum-Dot-Based Molecular Ruler for Multiplexed Optical Analysis
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DOI：
10.1002/anie.201002943
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发表时间：
2010-01-01
影响因子：
16.6
通讯作者：
Hildebrandt, Niko
中科院分区：
文献类型：
--
作者：
Morgner, Frank;Geissler, Daniel;Hildebrandt, Niko
基于Förster共振能量转移（FRET）的应用在生物学、生物化学、医学和其他生命科学领域的体外和体内纳米尺度系统内的浓度和距离测定中发挥着重要作用。[1-3]由于FRET的距离依赖性，分子系统在1 - 10 nm范围内的结构变化可以远低于光衍射极限的高精度测量。Stryer等人[4，5]在40多年前证明了光谱尺FRET技术，并且它仍然经常用于通过光谱学和显微镜进行分子间和分子内相互作用的体内和体外研究，直到单分子水平。[6-9]已经开发了几种基于FRET的用于功能性细胞内研究的生物传感器。[10 - 14]虽然这些应用中的大多数使用单个传感器，但是最近已经有一些使用荧光蛋白的双FRET对用于细胞成像的发展，[15 - 17]甚至具有单个激发波长。[18]使用多重FRET技术允许同时测量多个距离或构象变化，从而减少时间和精力，同时由于同时事件的可能相关性而增加生物分析信息。荧光铽配合物（LTCs）作为供体和半导体量子点（QD）作为受体的FRET对组合具有显着的优势，灵敏度，距离和多参数分析相比，其他供体-受体对。[19由于大的重叠积分值，可以实现高达11 nm的异常长的Förster半径（R0，FRET效率为50%的供体-受体距离），而传统的供体-受体对具有小得多的R0值，很少超过6 nm。[24]虽然已经开发了纳米等离子体分子标尺，可以测量高达约70 nm的距离，[25，26]这些应用使用相对较大的贵金属纳米颗粒（高达40 nm），并且限制了其同时测量不同系统的可变距离的多路复用（例如，在一次测量中的几个不同的细胞内功能事件）。韦斯等人的开创性工作展示了使用量子点和超高分辨率共定位（UHRC）的多路复用光学标尺。[27]虽然FRET在分辨率精度和动态测量方面具有优势，[28] UHRC非常适合测量几纳米到几十微米范围内的距离。[29]使用QD进行细胞内研究的两个非常重要的方面是这些纳米传感器的形状和大小，这对于例如细胞渗透和评估纳米颗粒对靶生物分子的影响至关重要。用TEM测量半导体材料的核/壳尺寸是可能的，具有相对良好的精度。然而，设备是相当昂贵的，实验是耗时的，通常不是在相同的条件下进行的生物分析研究的兴趣（如活细胞成像）。此外，很难获得整个生物相容性QD（例如，包括聚合物涂层和生物功能化）的准确尺寸，并且TEM仅提供二维图像。对于其聚合物包被的Qdot，Invitrogen提供了通过HPLC上的尺寸排阻色谱法测量的流体动力学直径（表1），其中通过相对于蛋白质标准曲线的保留时间确定尺寸。虽然这是对整体尺寸的估计，但形状没有考虑在内。
Applications based on Förster resonance energy transfer (FRET) play an important role in the determination of concentrations and distances within nanometer-scale systems in vitro and in vivo in the fields of biology, biochemistry, medicine, and other life sciences.[1–3] Due to the rÀ6 distance dependence of FRET, structural changes of molecular systems in the 1–10 nm range can be measured with high accuracy far below the light diffraction limit. Stryer et al.[4, 5] demonstrated the spectroscopic ruler FRET technique more than 40 years ago, and it is still frequently used for in-and exvivo studies of inter-and intramolecular interactions by spectroscopy and microscopy down to the single-molecule level.[6–9] Several FRET-based biosensors for functional intracellular investigations have been developed.[10–14] Although most of these applications use single sensors, there have been some recent developments of dual FRET pairs for cellular imaging using fluorescent proteins,[15–17] and even with a single excitation wavelength.[18] Using a multiplexed FRET technique allows the simultaneous measurement of multiple distances or conformational changes, thereby decreasing time and effort whilst increasing bioanalytical information due to the possible correlation of simultaneous events. The FRET pair combination of luminescent terbium complexes (LTCs) as donors and semiconductor quantum dots (QDs) as acceptors holds significant advantages concerning sensitivity, distance, and multiparametric analysis compared to other donor–acceptor pairs.[19, 20] Due to large overlap integral values, exceptionally long Förster radii (R0, the donor–acceptor distance at which the FRET efficiency is 50%) of up to 11 nm can be achieved,[21–23] whereas conventional donor–acceptor pairs have much smaller R0 values that rarely exceed 6 nm.[24] Although nanoplasmonic molecular rulers have been developed for which distances of up to about 70 nm can be measured,[25, 26] these applications use relatively large noble metal nanoparticles (up to 40nm) and are restricted in their multiplexed use of simultaneously measuring variable distances of different systems (for example, several different intracellular functional events within one measurement). The pioneering work of Weiss et al. demonstrated multiplexed optical rulers using quantum dots and ultrahigh-resolution colocalization (UHRC).[27] Although FRET has advantages concerning resolution accuracy and dynamic measurements,[28] UHRC is well-suited to measuring distances in the range of few nanometers to tens of micrometers.[29]Two very important aspects for intracellular studies with QDs are the shape and the size of these nanosensors, which can be crucial, for example, for cell penetration and for evaluation of the nanoparticle impact on the targeted biomolecules. Measuring the core/shell dimensions of the semiconductor material with TEM is possible with relatively good accuracy. However, the equipment is rather expensive and the experiments are time-consuming and usually not performed under the same conditions as the bioanalytical studies of interest (such as live-cell imaging). Moreover, it is very difficult to acquire accurate dimensions for the entire biocompatible QD (for example including polymer coating and biofunctionalization), and TEM provides only twodimensional images. For their polymer coated Qdots, Invitrogen provides hydrodynamic diameters (Table 1) measured by size-exclusion chromatography on HPLC, where the sizes are determined by retention time relative to a standard curve of proteins. Although this is an estimation of the overall size, the shape is not taken into account …