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Ratiometric fluorescence spectra of Oregon Green-488 (peak ~525 nm) and Ru-DPP (peak ~610 nm) in the presence of three levels of oxygen in aqueous solution.
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The spectra on the left show an example of ratiometric oxygen sensing using PEBBLE fluorescence in solution. Oregon Green can be seen to stay constant as the Ruthenium dye changes its intensity in response to varying amounts of oxygen - from Nitrogen bubbled solution on the top (no oxygen) to air saturated water in the middle, to Oxygen saturated solution on the bottom.
Each of these peaks separately changes in intensity due to varying amounts of PEBBLEs present in the solution, or due to changes in the intensity of the excitation light. But, the ratio of the two peaks does not change with either of these things. So, because each PEBBLE contains both dyes, an internal reference for changes in excitation or PEBBLE concentration is always carried along with each of these nano-devices.
Plot showing BSA interference with free dyes, but not with PEBBLE Oxygen sensors.
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Another serious problem of free dyes which PEBBLEs avoid is interference by things like non-specific protein binding (which happens a lot in a cellular environment). This plot shows that the Ru/Oregon Green fluorescence ratio goes up with increasing amounts of Bovine Serum Albumin (BSA) present in the solution, while the PEBBLE response stays constant - which is the desired result since the amount of Oxygen in solution is not changing. This illustrates the way in which the PEBBLE matrix protects the dyes from their environment. The converse is also true: The PEBBLE matrix protects the cells from any potentially harmful chemicals used for sensing, so a much wider range of compounds can be used than with free dyes. |
