Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors
Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors
复制标题
DOI：
10.1021/ac990630n
复制
发表时间：
1999-11-01
影响因子：
7.4
通讯作者：
Philbert, MA
中科院分区：
文献类型：
--
作者：
Clark, HA;Kopelman, R;Philbert, MA
光学纳米传感器或 PEBBLE（通过生物局部嵌入封装的探针）已被生产用于测量细胞内 pH 值和钙。介绍并讨论了五种 pH 敏感传感器和三种不同的钙选择性传感器。每个传感器都结合了离子选择性荧光指示剂和捕获在丙烯酰胺聚合物基质内的离子不敏感内标，每个传感器都提供了校准和线性范围。掺入 PEBBLE 中的染料的光漂白作用与掺入基质中的相应游离染料的光漂白作用相当。这些 PEBBLE 传感器在许多测量中都是完全可逆的。 48 小时内，荧光指示剂从聚合物中的浸出率低于 50%（请注意，典型的应用时间仅为几个小时）。 PEBBLE 传感器还应用于线粒体通透性转变过程中神经细胞细胞质中钙通量的细胞内分析。具体来说，不同类型的细胞（星形胶质细胞与神经元衍生细胞）之间对有毒物质间二硝基苯（DNB）的反应存在明显差异。使用 PEBBLE 传感器可以定量区分人 SY5Y 神经母细胞瘤和 C6 神经胶质瘤响应 DNB 挑战的能力之间的细微差异。具体来说，已经实现了细胞内钙（细胞死亡前体）的测量。
Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective fluorescent indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix, Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of fluorescent indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Specifically, a distinct difference is noted between cells of different types (astrocyte vs neuron-derived cells) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle differences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB, Specifically, measurement of intracellular calcium, the precursor to cell death, has been achieved.