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Upconversion nanoparticles (UCNPs) can provide a vehicle for chemical imaging by coupling chemically sensitive dyes and quenchers. The mechanism for coupling of two anthraquinone dyes, Calcium Red and Alizarin Red S, was investigated as a function of pH. The green emission band of the UCNPs was quenched by a pH-dependent inner filter effect (IFE) while the red emission band remained unchanged and acted as the reference signal for ratiometric pH measurements. Contrary to previous expectation, there was little evidence for a resonance energy transfer (RET) mechanism even when the anthraquinones were attached onto the UCNPs through electrostatic attraction. Since the UCNPs are point emitters, only emitters close to the surface of the UCNP are within the expected Förster distance and UC-RET is <10%. The theoretical and experimental analysis of the interaction between UCNPs and pH-sensitive quenchers will allow the design of UCNP pH sensors for determination of pH via IFE. [ABSTRACT FROM AUTHOR]

Taking advantage of BRET, a mutant firefly luciferase with higher pH- and thermo-stability than the wild-type could be coupled with the red-emitting fluorescent protein of mCherry in both a fused and unfused format. The BRET pair allows >40% of the light emitted to be red shifted over 600 nm to the mCherry acceptor wavelength. Taking the expected quantum yield for mCherry (0.22), a good fit to predicted light transfer is shown, with no other losses. Two measurements are considered for ATP determination: (a) a ratiometric technique for ATP measurement using both donor and acceptor emission intensities, making the calibration slope independent of protein concentration in a broad range. This measurement was limited by the BRET efficiency and the low quantum yield of the mCherry acceptor, but this detection limit might be improved with other fluorescent proteins with higher quantum yield. The fused BRET pair also resulted in a small increase in the BRET ratio. (b) An ATP dependent shift in the wavelength maximum using just the acceptor mCherry emission was also proposed for ATP determination. This did not require a high BRET efficiency and only uses emission above 600 nm to obtain the acceptor emission maximum, but not its intensity; it is independent of protein concentration across a broad range. This offers a novel and robust method for determination of ATP between 10−11 to 10−5 M with an easy baseline calibration with ATP concentration >10−4 M. [ABSTRACT FROM AUTHOR]