Laser Processing and Plasmonics Laboratory, Department of Engineering Physics, Polytechnique Montréal, C.P. 6079, Succursale Centre-Ville, Montréal, QC H3C 3A7, Canada.
Nanoscale. 2016 Jul 21;8(27):13263-72. doi: 10.1039/c6nr01257d. Epub 2016 Jun 23.
Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K(+) channel subunit KV1.1) on human cancer CD44(+) EGFR(+) KV1.1(+) MDA-MB-231 cells and reference CD44(-) EGFR(-) KV1.1(+) 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence.
选择标记、鉴定和细胞表面生物标志物的空间分布可以提供重要的临床信息，例如区分健康细胞和患病细胞、疾病的演变以及选择最佳的患者特异性治疗方法。免疫荧光是检测细胞表达的生物标志物的金标准。然而，与荧光染料偶联的抗体（Abs）仍然受到其光漂白、对环境的高灵敏度、低光强度以及宽吸收和发射光谱的限制。免疫等离子体学是一种基于可视化针对细胞表面生物标志物的 Abs 功能化等离子体纳米粒子（fNPs）的新型显微镜方法。可调谐 fNPs 应该比免疫荧光提供更高的多重检测能力，因为 fNPs 随时间推移具有更好的光稳定性，在等离子体峰值波长强烈散射光，并且易于功能化。在本文中，我们通过免疫等离子体学方法实验证明了准确的多重检测。首先，我们实现了对人类癌症 CD44(+) EGFR(+) KV1.1(+) MDA-MB-231 细胞和参考 CD44(-) EGFR(-) KV1.1(+) 661W 细胞上的三种靶向细胞表面生物标志物（细胞分化抗原 44（CD44）、表皮生长因子受体（EGFR）和电压门控 K(+)通道亚基 KV1.1）的选择性标记。用三种稳定的特异性免疫等离子体标记物（功能化银纳米球（CD44-AgNSs）、金（Au）纳米球（EGFR-AuNSs）和金纳米棒（KV1.1-AuNRs））检测到的标记效率通过反射光显微镜（RLM）与免疫荧光相似。其次，我们引入了一种改进的方法，用于基于 RLM 的快速 z 扫描对 fNPs 进行 3D 定位和光谱识别，该方法使用三个光谱滤波器对应于细胞环境中免疫等离子体标记的等离子体峰值波长（500nm 用于 80nm AgNSs、580nm 用于 100nm AuNSs 和 700nm 用于 40nm×92nm AuNRs）。第三，所开发的技术简单且与生物和临床实验室中使用的标准 epi-荧光显微镜兼容。因此，3D 多重免疫等离子体显微镜已经准备好作为免疫荧光的低成本替代方案在临床应用中使用。
