Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States.
Anal Chem. 2012 Jan 17;84(2):978-86. doi: 10.1021/ac202521e. Epub 2012 Jan 5.
Ca(2+) is a universal second messenger and plays a major role in intracellular signaling, metabolism, and a wide range of cellular processes. To date, one of the most successful approaches for intracellular Ca(2+) measurement involves the introduction of optically sensitive Ca(2+) indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca(2+) indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most-sensitive indicators, cytotoxicity of the indicators, interference from nonspecific binding caused by cellular biomacromolecules, challenging calibration, and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca(2+) indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine-based Ca(2+) fluorescence indicators. The rhod-2-containing PEBBLEs presented here show a stable sensing range at near-neutral pH (pH 6-9). Because of the protection of the PEBBLE matrix, the interference of protein-nonspecific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (K(d) = 478 nM) and intracellular (K(d) = 293 nM) measurements. These nanosensors are useful quantitative tools for the measurement and imaging of the cytosolic nanomolar free Ca(2+) levels.
钙离子是一种普遍的第二信使，在细胞内信号转导、代谢和广泛的细胞过程中发挥着重要作用。迄今为止，用于细胞内钙离子测量的最成功方法之一是将对钙离子敏感的指示剂引入活细胞中，并结合数字成像显微镜。然而，使用游离钙离子指示剂进行细胞内传感和成像存在几个限制，例如最敏感的指示剂是非比率测量、指示剂的细胞毒性、细胞生物大分子引起的非特异性结合干扰、校准困难以及指示剂分子的非预期螯合。当钙离子指示剂被包裹在多孔惰性聚丙烯酰胺纳米粒子中时，这些问题可以最小化。我们提出了用罗丹明基钙离子荧光指示剂封装的 PEBBLE 纳米传感器。这里介绍的含有 rhod-2 的 PEBBLE 具有近中性 pH 值（6-9）的稳定传感范围。由于 PEBBLE 基质的保护，指示剂与蛋白质的非特异性结合的干扰最小。 rhod-2 的 PEBBLE 在溶液内（K(d) = 478 nM）和细胞内（K(d) = 293 nM）测量中都具有纳摩尔动态传感范围。这些纳米传感器是测量和成像细胞溶质纳摩尔游离钙离子水平的有用定量工具。
