Xu H, Aylott J W, Kopelman R, Miller T J, Philbert M A
Department of Chemistry, University of Michigan, Ann Arbor 48109-1055, USA.
Anal Chem. 2001 Sep 1;73(17):4124-33. doi: 10.1021/ac0102718.
The first sol-gel-based, ratiometric, optical nanosensors, or sol-gel probes encapsulated by biologically localized embedding (PEBBLEs), are made and demonstrated here to enable reliable, real-time measurements of subcellular molecular oxygen. Sensors were made using a modified Stöber method, with poly(ethylene glycol) as a steric stabilizer. The radii of these spherical PEBBLE sensors range from about 50 to 300 nm. These sensors incorporate an oxygen-sensitive fluorescent indicator, Ru(II)-tris(4,7-diphenyl-1,10-phenanthroline) chloride ([Ru(dpp)3]2+), and an oxygen-insensitive fluorescent dye, Oregon Green 488-dextran, as a reference for the purpose of ratiometric intensity measurements. The PEBBLE sensors have excellent reversibility, dynamic range, and stability to leaching and photobleaching. The small size and inert matrix of these sensors allow them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. Applications of sol-gel PEBBLEs inserted in rat C6 glioma cells for real-time intracellular oxygen analysis are demonstrated. Compared to using free dyes for intracellular measurements, the PEBBLE matrix protects the fluorescent dyes from interference by proteins in cells, enabling reliable in vivo chemical analysis. Conversely, the matrix also significantly reduces the toxicity of the indicator and reference dyes to the cells, so that a wide variety of dyes can be used in optimal fashion.
本文制备并展示了首批基于溶胶 - 凝胶的比率型光学纳米传感器，即生物定位包埋封装的溶胶 - 凝胶探针（PEBBLEs），以实现对亚细胞分子氧的可靠实时测量。传感器采用改良的斯托伯方法制备，使用聚乙二醇作为空间稳定剂。这些球形PEBBLE传感器的半径范围约为50至300纳米。这些传感器包含一种对氧敏感的荧光指示剂氯化钌（II） - 三（4,7 - 二苯基 - 1,10 - 菲咯啉）（[Ru(dpp)3]2+）和一种对氧不敏感的荧光染料俄勒冈绿488 - 葡聚糖，用于比率强度测量的参考。PEBBLE传感器具有出色的可逆性、动态范围以及对浸出和光漂白的稳定性。这些传感器的小尺寸和惰性基质使其能够插入活细胞中，对其生物学功能产生最小的物理和化学扰动。展示了将溶胶 - 凝胶PEBBLEs插入大鼠C6胶质瘤细胞中进行实时细胞内氧分析的应用。与使用游离染料进行细胞内测量相比，PEBBLE基质可保护荧光染料免受细胞内蛋白质的干扰，从而实现可靠的体内化学分析。相反，该基质还显著降低了指示剂和参考染料对细胞的毒性，因此可以以最佳方式使用多种染料。
