Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors

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.