Tavernaro Isabella, Cavelius Christian, Peuschel Henrike, Kraegeloh Annette
INM - Leibniz Institute for New Materials, Nano Cell Interactions Group, Campus D2 2, D-66123 Saarbrücken, Germany.
nanoSaar Lab GmbH, Comotorstr. 2, D-66802 Überherrn, Germany.
Beilstein J Nanotechnol. 2017 Jun 21;8:1283-1296. doi: 10.3762/bjnano.8.130. eCollection 2017.
In recent years, fluorescent nanomaterials have gained high relevance in biological applications as probes for various fluorescence-based spectroscopy and imaging techniques. Among these materials, dye-doped silica nanoparticles have demonstrated a high potential to overcome the limitations presented by conventional organic dyes such as high photobleaching, low stability and limited fluorescence intensity. In the present work we describe an effective approach for the preparation of fluorescent silica nanoparticles in the size range between 15 and 80 nm based on L-arginine-controlled hydrolysis of tetraethoxysilane in a biphasic cyclohexane-water system. Commercially available far-red fluorescent dyes (Atto647N, Abberior STAR 635, Dy-647, Dy-648 and Dy-649) were embedded covalently into the particle matrix, which was achieved by aminosilane coupling. The physical particle attributes (particle size, dispersion, degree of agglomeration and stability) and the fluorescence properties of the obtained particles were compared to particles from commonly known synthesis methods. As a result, the spectroscopic characteristics of the presented monodisperse dye-doped silica nanoparticles were similar to those of the free uncoupled dyes, but indicate a much higher photostability and brightness. As revealed by dynamic light scattering and ζ-potential measurements, all particle suspensions were stable in water and cell culture medium. In addition, uptake studies on A549 cells were performed, using confocal and stimulated emission depletion (STED) microscopy. Our approach allows for a step-by-step formation of dye-doped silica nanoparticles in the form of dye-incorporated spheres, which can be used as versatile fluorescent probes in confocal and STED imaging.
近年来，荧光纳米材料在生物应用中作为各种基于荧光的光谱学和成像技术的探针变得高度相关。在这些材料中，染料掺杂的二氧化硅纳米颗粒已显示出克服传统有机染料所呈现的局限性的巨大潜力，如高光漂白、低稳定性和有限的荧光强度。在本工作中，我们描述了一种基于L-精氨酸控制四乙氧基硅烷在双相环己烷-水体系中水解来制备尺寸在15至80纳米之间的荧光二氧化硅纳米颗粒的有效方法。市售的远红荧光染料（Atto647N、Abberior STAR 635、Dy-647、Dy-648和Dy-649）通过氨基硅烷偶联共价嵌入颗粒基质中。将所得颗粒的物理属性（粒径、分散性、团聚程度和稳定性）和荧光特性与来自常见合成方法的颗粒进行了比较。结果表明，所呈现的单分散染料掺杂二氧化硅纳米颗粒的光谱特性与游离未偶联染料的相似，但显示出更高的光稳定性和亮度。动态光散射和ζ电位测量表明，所有颗粒悬浮液在水和细胞培养基中均稳定。此外，使用共聚焦和受激发射损耗（STED）显微镜对A549细胞进行了摄取研究。我们的方法允许以染料掺入球体的形式逐步形成染料掺杂的二氧化硅纳米颗粒，其可作为共聚焦和STED成像中的通用荧光探针。
