Ioannou Dimitris, Tempest Helen G, Skinner Benjamin M, Thornhill Alan R, Ellis Michael, Griffin Darren K
Department of Biosciences, University of Kent, Canterbury, UK.
Chromosome Res. 2009;17(4):519-30. doi: 10.1007/s10577-009-9051-0. Epub 2009 Jul 31.
In the field of nanotechnology, quantum dots (QDs) are a novel class of inorganic fluorochromes composed of nanometre-scale crystals made of a semiconductor material. Given the remarkable optical properties that they possess, they have been proposed as an ideal material for use in fluorescent in-situ hybridization (FISH). That is, they are resistant to photobleaching and they excite at a wide range of wavelengths but emit light in a very narrow band that can be controlled by particle size and thus have the potential for multiplexing experiments. The principal aim of this study was to compare the potential of QDs against traditional organic fluorochromes in both indirect (i.e. QD-conjugated streptavidin) and direct (i.e. synthesis of QD-labelled FISH probes) detection methods. In general, the indirect experiments met with a degree of success, with FISH applications demonstrated for chromosome painting, BAC mapping and use of oligonucleotide probes on human and avian chromosomes/nuclei. Many of the reported properties of QDs (e.g. brightness, 'blinking' and resistance to photobleaching) were observed. On the other hand, signals were more frequently observed where the chromatin was less condensed (e.g. around the periphery of the chromosome or in the interphase nucleus) and significant bleed-through to other filters was apparent (despite the reported narrow emission spectra). Most importantly, experimental success was intermittent (sometimes even in identical, parallel experiments) making attempts to improve reliability difficult. Experimentation with direct labelling showed evidence of the generation of QD-DNA constructs but no successful FISH experiments. We conclude that QDs are not, in their current form, suitable materials for FISH because of the lack of reproducibility of the experiments; we speculate why this might be the case and look forward to the possibility of nanotechnology forming the basis of future molecular cytogenetic applications.
在纳米技术领域，量子点（QDs）是一类新型的无机荧光染料，由半导体材料制成的纳米级晶体组成。鉴于它们所具有的卓越光学特性，它们被提议作为荧光原位杂交（FISH）的理想材料。也就是说，它们抗光漂白，能在很宽的波长范围内激发，但发射的光带宽很窄，可通过颗粒大小控制，因此有进行多重实验的潜力。本研究的主要目的是在间接（即量子点偶联链霉亲和素）和直接（即合成量子点标记的FISH探针）检测方法中，比较量子点与传统有机荧光染料的潜力。总体而言，间接实验取得了一定程度的成功，已证明FISH可用于染色体描绘、BAC定位以及在人类和禽类染色体/细胞核上使用寡核苷酸探针。观察到了许多报道的量子点特性（如亮度、“闪烁”和抗光漂白）。另一方面，在染色质凝聚程度较低的区域（如染色体周边或间期核内）更频繁地观察到信号，并且明显存在显著的信号渗漏到其他滤光片的情况（尽管报道的发射光谱很窄）。最重要的是，实验成功具有间歇性（有时即使在相同的平行实验中也是如此），这使得提高可靠性的尝试变得困难。直接标记实验显示有量子点 - DNA构建体生成的证据，但没有成功的FISH实验。我们得出结论，就目前的形式而言，量子点不是适用于FISH的材料，因为实验缺乏可重复性；我们推测了这种情况可能的原因，并期待纳米技术成为未来分子细胞遗传学应用基础的可能性。
