| The focus of this work has been the production, characterization and application of nanometer-sized optical sensors to chemical analysis inside single living cells. The work began with the application of optical fiber based sensors that had be pulled into a submicron tip and coated with a polymer containing the active components. The development of a submicron optical fiber "supertip" facilitated insertion of these sensors into cells. A potassium selective sensor was used to monitor the effect of an ion channel-blocking agent inside a single mouse oocyte.; In order to circumvent the size constraints posed by the fiber optic based sensors, spherical optical nanosensors, or PEBBLEs (Probes Encapsulated Biologically Localized Embedding), have been produced in sizes including 20 and 200 nm in diameter. These sensors are fabricated in a microemulsion for which a generalized polymerization method has been developed, and consist of fluorescent indicators and an ion insensitive internal standard entrapped in a polyacrylamide matrix. The PEBBLE matrix protects the fluorescent dye from interference by proteins. Sensor response times are less than one millisecond. Five varieties of pH sensitive sensors and three different calcium selective sensors have been characterized. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye. The leaching of fluorescent indicator from the polymer is less than 50% over a 48 hour period. Cell viability assays indicate that the PEBBLEs are biocompatible. Several methods for delivering sensors to the cytoplasm of single cells have been studied, including liposomal delivery, gene gun bombardment and pico-injection into single living cells.; The PEBBLE sensors have 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. |
