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.