Cu+- and Cu2+-sensitive PEBBLE fluorescent nanosensors using DsRed as the recognition element

The red fluorescent protein (DsRed) has typically been used as either a fluorescent tag or FRET acceptor, but here, we detail its use as a recognition element for Cu2+ and Cu+ in a fluorescent photonic explorer for bioanalysis with biologically localized embedding (PEBBLE) nanosensor. DsRed and the reference dye Alexa Fluor 488 have been encapsulated within a polyacrylamide matrix by a microemulsion polymerization process, to produce spherical, ratiometric, 80 nm-sized sensors, which allow for spatially resolved real-time measurements. These nanobiosensors have excellent selectivity and sensitivity towards copper ions, with a detectable range in the nanomolar (ppb) regime, even in the presence of other divalent and heavy metal ions. The nanosensors are both photostable and reversible, which allows for continuous monitoring. (c) 2005 Elsevier B.V. All rights reserved.