Mapping Local pH in Live Cells Using Encapsulated Fluorescent SERS Nanotags
Mapping Local pH in Live Cells Using Encapsulated Fluorescent SERS Nanotags
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DOI：
10.1002/smll.200901893
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发表时间：
2010-03-08
期刊：
影响因子：
13.3
通讯作者：
Moskovits, Martin
中科院分区：
文献类型：
--
作者：
Pallaoro, Alessia;Braun, Gary B.;Moskovits, Martin
了解细胞对纳米材料的摄取和处理对治疗学和诊断学具有重要意义。尽管纳米粒子制剂已被开发为已被证实的成像剂和有希望的药物输送载体，但关于它们作为局部环境传感器的内在化仍有许多需要了解，即使是在纳米尺度上测定pH的相对简单的情况下也是如此。据报道，体外细胞培养的策略是使用带有阳离子试剂的含有货物或染料标记的纳米颗粒，阳离子通过静电相互作用促进与细胞膜的结合，诱导细胞膜包裹颗粒，然后颗粒通过内吞作用内化。[1-3]无法通过主动运输或扩散穿过细胞膜的营养物质和分子以及大于几纳米的无机颗粒使用这种路线。这一复杂的过程还被用于吸收核酸、药物和多肽，以及用于药物输送、传感和生物相容性应用的聚合物。虽然已经报道了在pH敏感的双色荧光聚合物内吞后监测细胞内部pH的有希望的努力，但仍然存在光稳定性和波长优化的问题。理想的纳米探针应该是光稳定的、灵敏的、可激发的，在近红外(NIR)波长下，细胞和组织多少是透明的，自体荧光是最小的。一系列基于荧光的材料已被开发为成像剂和局部探针，用于检测特定生物标志物的存在或局部环境条件。例如，pH敏感的荧光探针已被用来通过比较两个荧光带之间的强度比来确定内体随时间变化的酸性[6-8]。[4，5，9]这种应用突出了测量强度比的改进方法的必要性；类似地，在原位同时测量多个生物标志物可能是未来医学分析和诊断的重要组成部分。由于大多数荧光带的宽度和无特征，在这种多路应用中使用荧光通常不是一个好的信号。此外，许多发光体在重复或强烈的光照下漂白。基于表面增强拉曼光谱(SERS)的pH传感[10-12]已经被探索作为荧光的替代应用，在应用中遇到了上述挑战。除了由于SERS波段狭窄而具有更大的多路复用潜力外，SERS还可以常规地由近红外波长激发，从而提高光稳定性，潜在地将纳米粒子跟踪的时间窗口从几分钟延长到几个小时。这些特性使SERS成为一种吸引人的成像技术和生物功能的探针。[13，14]众所周知，最明亮的SERS信号来自金属纳米颗粒二聚体和小团簇，而不是单个纳米颗粒，或者来自纳米工程的纳米结构，如纳米贝壳。可靠的表面增强拉曼散射的一个主要障碍是开发稳定、可重现和明亮的表面增强拉曼散射活性纳米颗粒。在本通信中，我们使用预连接并随后包覆的银纳米颗粒中包含的4-巯基苯甲酸(MBA)作为pH敏感探针。纳米颗粒簇被包裹并与蛋白质货物结合，并被功能化以供细胞摄取。[15]我们通过跟踪涂层的荧光，同时使用…测量内体的局部pH，证明了这些优化的表面增强拉曼散射纳米探针与荧光成像是兼容的
Understanding uptake and processing of nanomaterials by cells has implications for therapeutics and diagnostics. Although nanoparticle agents have been developed as proven imaging agents and promising drug-delivery vehicles, much remains to be learned regarding their internalization as local environmental sensors, even for the relatively simple case of pH determination at the nanoscale. Strategies for in vitro cell culture have been reported that use cargo-containing or dyelabeled nanoparticles with a cationic agent, which promotes binding to the cell membrane through an electrostatic interaction, inducing the cell membrane to wrap around the particle, which is then internalized through endocytosis.[1–3] Nutrients and molecules that are not able to cross the membrane through active transport or diffusion and inorganic particles bigger than a few nanometers use this route. This complex process is also exploited in the uptake of nucleic acids, drugs and peptides, and of polymers for drug delivery, sensing and biocompatibility applications. Although promising efforts to monitor the interior pH in a cell after endocytosis of pH-sensitive dual-color ffuorescent polymers have been reported,[4, 5] issues of photostability and wavelength optimization remain. An ideal nanoprobe would be photostable, sensitive, and excitable with near-infrared (NIR) wavelengths at which cells and tissues are somewhat transparent and autoffuorescence is minimized. A wide range of ffuorescence-based materials have been developed as imaging agents and as local probes of the presence of specific biomarkers or of local environmental conditions. For example pH-sensitive ffuorescence probes have been used to determine the time-dependent acidity of endosomes [6–8] through a comparison of the intensity ratio between two ffuorescence bands.[4, 5, 9] Such applications highlight the need for improved methods to measure intensity ratios; similarly, the simultaneous measurement of multiple biomarkers in situ is likely to be an important component of future medical analysis and diagnostics. Fluorescence is normally not a good signal to use in such multiplex applications on account of the breadth and featurelessness of most ffuorescence bands. Moreover, many ffuorophores bleach under repeated or intense illumination. Surface-enhance Raman spectroscopy (SERS)-based pH sensing has been explored [10–12] as an alternative to ffuorescence for application in which one encounters the above challenges. In addition to the greater potential for multiplexing on account of the narrowness of SERS bands, SERS can be routinely excited by NIR wavelengths leading to increased photostability, potentially prolonging the time window for nanoparticle tracking from minutes to hours. These characteristics make SERS an appealing technique both for imaging and as a probe for biological function.[13, 14] It is well known that the brightest SERS signals originate from metal nanoparticle dimers and small clusters rather than single nanoparticles, or from nanoengineered nanostructures such as nanoshells. A major hurdle for reliable SERS is the development of stable, reproducible, and bright SERS-active nanoparticles. In this Communication, we use 4-mercaptobenzioc acid (MBA) contained within pre-linked and subsequently coated Ag nanoparticles as a pH-sensitive probe. The nanoparticle cluster is encapsulated and bound with protein cargo and functionalized for cellular uptake.[15] We demonstrate that these optimized SERS nanoprobes are compatible with ffuorescent imaging by tracking the ffuorescence from the coating, while measuring the local pH of the endosomes using …