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 locali zed embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calciu m-selective sensors are presented and discussed. Each sensor combines an io n-selective fluorescent indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix, Calibrations and linear ra nges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is inco rporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of fluorescent indicator from the polymer i s 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 intracellu lar analysis of the calcium flux in the cytoplasm of neural cells during th e mitochondrial permeability transition. Specifically, a distinct differenc e is noted between cells of different types (astrocyte vs neuron-derived ce lls) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle di fferences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB, Specifically, measurement of intracellular c alcium, the precursor to cell death, has been achieved.