A real-time ratiometric method for the determination of molecular oxygen inside living cells using sol-gel-based spherical optical nanosensors with applications to rat C6 glioma
A real-time ratiometric method for the determination of molecular oxygen inside living cells using sol-gel-based spherical optical nanosensors with applications to rat C6 glioma
复制标题
DOI：
10.1021/ac0102718
复制
发表时间：
2001-09-01
影响因子：
7.4
通讯作者：
Philbert, MA
中科院分区：
文献类型：
--
作者：
Xu, H;Aylott, JW;Philbert, MA
第一个基于溶胶-凝胶、比率、光学纳米传感器或生物局部包埋(鹅卵石)封装的溶胶-凝胶探针，在这里被制造和展示，以实现对亚细胞分子氧的可靠、实时测量。传感器采用改进的斯托伯法，以聚乙二醇作空间稳定剂。这些球形鹅卵石传感器的半径从50到300mN不等。这些传感器包括氧敏感的荧光指示剂Ru(Il)三(4，7-二苯基-1，10-菲咯啉)氯([Ru(DPP)(3)](2+))和氧不敏感的荧光染料俄勒冈绿488-葡聚糖，作为比率强度测量的参考。Pebble传感器具有良好的可逆性、动态范围和对浸出和光漂白的稳定性。这些传感器的小尺寸和惰性矩阵允许它们被插入活细胞，对其生物功能的物理和化学干扰最小。在大鼠C6胶质瘤细胞中插入的溶胶-凝胶鹅卵石用于实时细胞内氧分析的应用被证明。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，基质还显著降低了指示剂和参比染料对细胞的毒性，因此各种染料都可以以最佳方式使用。
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 Stober method, with poly(ethylene glycol) as a steric stabilizer. The radii of these spherical PEBBLE sensors range from about 50 to 300 mn. These sensors incorporate an oxygen-sensitive fluorescent indicator, Ru(Il)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.