Small nanoparticles: Design and synthesis of shape variants as multiplex biological probes for immuno-electron microscopy.

电子显微镜(EM)是细胞生物学研究中的一项核心技术，可以对细胞结构进行纳米(Nm)尺度的分析。为了显示特定蛋白质和脂类的精确位置，使用了结合到抗体上的电子密度标记。金纳米粒子由于其尺寸在2-15 nm范围内容易调节以及与靶标抗体的简单结合而成为首选。通常，一个样本内的标记(免疫-EM)被限制在三个靶标上，使用视觉上可分离的黄金尺寸(5-、10-和15 nm)。越来越多的细胞生物学研究需要同时分析信令网络中更多的成分。此外，某些分析方法仅限于在非常窄的尺寸范围内使用黄金，进一步减少了标签选项。我们的目标是调整合成金色形状变体(条形、立方形、三角形、八面体)的方法，以生产适合免疫-EM的尺寸范围的颗粒。对于这些小尺寸(2-15 nm)的球形颗粒是默认的，通过增加视觉可识别的形状套件，我们将显著增加免疫-EM的标记选择。使用我们实验室开发的技术，这些颗粒可以与几乎任何化合物不可逆转地联系在一起。我们将把纳米颗粒与针对调节细胞生长和存活的重要途径--RAS级联反应的组件的抗体结合起来。这些成分定位于细胞表面，并可以使用我们实验室为研究分离的细胞表面碎片中的蛋白质和脂肪分布而开发的EM程序进行可视化。不幸的是，这种方法仅限于使用非常小的金(5 nm或更小)，因此形状变体的发展极大地增加了可以在单个样品中同时识别的成分的数量。我们的研究目的是使用这些工具来确定信号通路的组件被划分的程度，即在有助于提高信号效率或特异性的离散斑块中找到的程度。RAS是研究细胞信号通路中异构体特异性的模型蛋白质，与我们的EM方法相结合，提供了对细胞表面区划的见解，因此这项研究具有超越RAS生物学直接领域的意义。在更广泛的意义上，新的形状变体将使目前依赖免疫-EM进行研究的整个细胞生物学社区受益。

Summary Electron microscopy (EM) is a core technique within cell biology research allowing nanometer (nm) scale analysis of cell structure. In order to visualise the precise location of specific proteins and lipids, electron dense labels conjugated to antibodies are used. Gold nanoparticles are preferred because of their easily tunable size in the range of 2-15 nm and simple conjugation to targetting antibodies. Typically, labelling within a sample (immuno-EM) is restricted to three targets using visually separable gold sizes (5-, 10- and 15 nm). Increasingly, cell biology research requires simultaneous analysis of many more components within for example signalling networks. In addition, certain analysis methods are limited to using gold within a very narrow size range, further reducing labelling options. We aim to adapt methods for synthesising gold shape variants (bars, cubes, triangular plates, octahedrons) to produce particles with a size range suitable for immuno-EM. At these small sizes (2-15 nm) spherical particles are the default, by increasing the suite of visually identifiable shapes we will significantly increase the labelling options for immuno-EM. Using technology developed in our laboratory, the particles can be irreversibly linked to almost any compound. We will bind the nanoparticles to antibodies targetted against components of an important pathway involved in regulating cell growth and survival - the Ras cascade. These components are localised on the cell surface and can be visualised using EM protocols developed in our laboratory for studying protein and lipid distributions within isolated cell surface fragments. Unfortunately, this approach is limited to using very small gold (5 nm or less), therefore the development of shape variants dramatically increases the number of components that can be simultaneously identified within a single sample. Our study aims use these tools to determine the extent to which components of the signalling pathway are compartmentalised i.e. found in discrete patches that help to improve signalling efficiency or specificity. Ras is a model protein for studying isoform specificity within cell signalling pathways and combined with our EM approach has provided insights into compartmentalisation of the cell surface, therefore the research has implications beyond the immediate field of Ras biology. In a broader sense, the new shape variants will benefit the entire cell biology community who currently rely on immuno-EM for their studies.