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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]

An enzyme-linked analytical nanosphere sensor (ANSor) is described, responding to enzyme-substrate turnover in the vicinity of a quantum dot (QD) due to cohnmobilized enzyme and pH sensitive ligand. QD capping by mercapto-alkanoic acids were rejected as a pH sensitive ligand, but with the use of a layer-by-layer assembly on mercaptopropionic capped QDs and an intermediate poly(allylamine hydrochloride) layer, anthraquinone sulfonate (calcium red, CaR) was introduced to modify the pH in the immobilized system > 8. QD-CaR absorption shows speciral overlap with QDroo emission at all pHs and gives a complex pH dependent fluorescence resonance energy transfer (FREL) efficiency, due to excited state proton transfer (χex = 540 nm; χem = 585 nm). In contrast QD615-CaR with spectral overlap between the QD and CaR gave a strong and reproducible pH response. QD-urease and QD-creatinine deiminase conjugates could be linked with pH changes produced by enzyme degradation of urea and crealinine, respectively. Close coupling between the pH sensitive QD and enzyme conjugate maximized signal compared with solution based assays: QD-urease and QD-CD bioconjugates were tested in model biological media (Dulbecco's modified Eagle's Medium and fetal calf serum) and in urine, showing a response in 3-4 min. [ABSTRACT FROM AUTHOR]

A new fluorescent nanosensor family for Zn2+ determination is reported based on azamacrocycle derivatization of CdSe/ZnS core/shell quantum dot nanoparticles. They are the first zinc ion sensors using QD nanoparticles in a host-guest and receptor-fluorophore system. Three azamacrocycles are demonstrated as receptors: TACN (1,4,7-triazacyclononane), cyclen (1,4,7,10-tetraazacyclododecane), and cyclam (1,4,8,11-tetraazacyclotetradecane). Azamacrocycles conjugated to QDs via an amide link interact directly with one of the photoinduced QD charge carriers, probably transferring the hole in the QD to the azamacrocycle, thereby disrupting the radiative recombination process. When zinc ion enters the azacrown, the lone pair electrons of the nitrogen atom become involved in the coordination and the energy level is no longer available for the hole-transfer mechanism, switching on the QD emission and a dramatic increase of the fluorescence intensity results, allowing the detection of low concentrations of zinc ions. Using this operating principle, three zinc ion sensors based on CdSe-ZnS core-shell QD nanoparticles showed a very good linearity in the range 5-500 μM, with detection limits lower than 2.4 μM and RSDs ∼3% (n = 10). In addition, the versatility of the sensors was demonstrated, since different sizes (and colors) of QDs can be employed and will respond to zinc in a similar way. In a study of interferences, the zinc-sensitive QDs showed good selectivity in comparison with other physiologically important cations and other transition metals tested. The results from fetal calf serum and samples mimicking physiological conditions suggested very good applicability in the determination of zinc ion in physiological samples. [ABSTRACT FROM AUTHOR]

The first CdSe/ZnS quantum dot photoluminescence lifetime-based pH nanosensor has been developed. The average lifetime of mercaptopropionic acid-capped QD nanosensors showed a linear response in the pH range of 5.2–6.9. These nanosensors have been satisfactorily applied for pH estimation in simulated intracellular media, with high sensitivity and high selectivity toward most of the intracellular components. [ABSTRACT FROM AUTHOR]