Experimental Procedure

An application that is truly unique to nanosensors involves monitoring living single cells in vivo [18-23]. Examples of this application in molecular biology will be discussed later. This section provides a description of the procedures for growing cell cultures for analysis using the nanosensors. Cell cultures were grown in a water-jacketed cell culture incubator at 37 °C in an atmosphere of 5% CO2 in air. Clone 9 cells, a rat liver epithelial cell line, were grown in Ham's F-12 medium (Gibco), supplemented with 10% fetal bovine serum and an additional 1 mM glutamine (Gibco). In preparation for an experiment, 1 x 105 cells in 5 mL of medium were seeded into standard dishes (Corning Costar Corporation). The growth of the cells was monitored daily by microscopic observation, and when the cells reached a state of confluence of 50-60%, the analyte solution was added and left in contact with the cells for 18 h (i.e., overnight). This procedure is designed to incubate the cells with the analyte molecules for subsequent monitoring using the nanosensors. The growth conditions were chosen so that the cells would be in log-phase growth during the chemical treatment, but would not be so close to confluence that a confluent mono-layer would form by the termination of the chemical exposure. The analyte solution was prepared as a 1 mM stock solution in reagent grade methanol, and further diluted in reagent grade ethanol (95%) prior to addition to the cells. Following chemical treatment, the medium containing the analyte was aspirated and replaced with standard growth medium, prior to the nanoprobe procedure.

Monitoring target analyte molecules in single cells was then performed using antibody nanoprobes in the following way. A culture dish of cells was placed on the prewarmed microscope stage, and the nanoprobe, mounted on the micropipette holder, was moved into position (i.e., in the same plane of the cells), using bright field microscopic illumination, so that the tip was outside the cell to be probed. The total magnification was usually 400 x. Under no room light and no microscope illumination, the laser shutter was opened to illuminate the optical fiber for excitation of the analyte molecules bound on the antibodies at the fiber tip. Usually, if the silver coating on the nanoprobe was appropriate, no light leaked out of the sidewall of the tapered fiber. Only a faint glow of laser excitation at the tip could be observed on the nanoprobe. A reading was first taken with the nanoprobe outside the cell and the laser shutter closed. The nanoprobe was then moved into the cell, inside the cell membrane, and extending into the cellular compartments of interest. The laser was again opened, and readings were then taken and recorded as a function of time during which the nanoprobe was inside the cell.

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