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ABSTRACT: A ratiometric Eu(III) complex has been developed that localises selectively within the endoplasmic reticulum of living cells. Careful calibration, using a time-gated spectral imaging microscope, allows the intensity ratio of emission bands and the variation of excited state lifetime to be used for pH determination, with a pKa of 7.15.
Chemical Communications 05/2013; · 6.17 Impact Factor
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James W Walton,
Adrien Bourdolle,
Stephen J Butler,
Marine Soulie,
Martina Delbianco,
Brian K McMahon, Robert Pal,
Horst Puschmann,
Jurriaan M Zwier,
Laurent Lamarque,
Olivier Maury,
Chantal Andraud,
David Parker
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ABSTRACT: The synthesis, structure and photophysical properties of a series of highly emissive europium complexes is reported. Certain complexes enter mammalian cells by macropinocytosis and stain the mitochondria selectively, allowing observation of the Eu emission in cellulo by time-gated spectral imaging.
Chemical Communications 01/2013; · 6.17 Impact Factor
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ABSTRACT: A series of Eu and Tb complexes of four different chiral ligands incorporating an azaxanthone sensitiser has been evaluated as probes for the bicarbonate anion. Their binding affinities were assessed at ambient pH with bicarbonate, lactate, citrate, phosphate and serum albumin. Binding was signalled by modulation of circularly polarised luminescence and apparent affinity constants were measured by examining changes in emission intensity ratios. Competition experiments show that with these species and ATP present at normal physiological values, bicarbonate can be determined selectively over the concentration range 10 to 35 mM. Bicarbonate levels are also reported by using a mixture of Eu and Tb complexes of a common ligand, examining the ratio of red/green emitted light. These methods have been adapted for the determination of bicarbonate in human serum and used for the assessment of mitochondrial levels of bicarbonate in several different cell types with confocal microscopy.
Chemistry 08/2012; 18(37):11604-13. · 5.93 Impact Factor
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ABSTRACT: Europium and terbium complexes of two structurally related ligands have been evaluated as optical probes to monitor changes in lysosomal pH; calibration using ionophores and fluorescent probes allows monitoring of the time dependence of lysosomal pH change, examining the green/red intensity ratio from internalised Tb-Eu complexes.
Chemical Communications 07/2012; 48(68):8520-2. · 6.17 Impact Factor
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ABSTRACT: Image and spectral intensity from bicarbonate-selective europium(III) probes localised in the mitochondria of cells is modulated reversibly by variation of external pCO(2), and is suppressed by addition of the carbonic anhydrase inhibitor, acetazolomide.
Chemical Communications 07/2011; 47(26):7347-9. · 6.17 Impact Factor
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ABSTRACT: This work aims to develop and validate a rapid analytical method that enables the measurement of citrate in seminal fluid samples. Samples were obtained from men who were within 9 weeks of a vasectomy operation. Two age ranges were examined, between 40-43 and 50-53 years old, with nearly 100 samples in each case. No patient clinical history was available for this anonymous study, simulating a random screening cohort. The concentration of citrate in 0.5 μL seminal fluid samples was assessed, using a europium emission luminescence method. This involves the ratiometric analysis of two well-separated europium(III) emission bands. Spectral data were obtained using a time-gated spectrometer whose construction and modification is described. Citrate values were confirmed by independent measurements using a citrate lyase enzymatic assay and by 700 MHz ¹H NMR analysis of the seminal fluid. Citrate concentrations were not statistically different between age groups and averaged 35.0(±14.6) mM for the 40-43 group, and 28.2(±12.7) mM for the 50-53 cohort; in each case a polymodal distribution was observed.
Journal of pharmaceutical and biomedical analysis 05/2011; 56(2):352-8. · 2.45 Impact Factor
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ABSTRACT: A terbium complex has been designed that may be used to sensitise formation of singlet oxygen (phi(em) 12%), allowing the creation of a ratiometric sensory system for pO(2) (versus the insensitive Eu analogue) and the definition of a reactive probe that can be used to cause local damage to cells loaded with the complex and exposed to laser excitation.
Chemical Communications 12/2009; · 6.17 Impact Factor
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ABSTRACT: Ratiometric methods of analysis have been developed for the selective determination of lactate or citrate in microlitre samples of human serum, urine or prostate fluids following comparison of anion binding affinities for a family of nine luminescent europium(III) complexes.
Organic & Biomolecular Chemistry 05/2009; 7(8):1525-8. · 3.70 Impact Factor
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ABSTRACT: To understand better the structure and function of biological systems, cell biologists and biochemists would like to have methods that minimally perturb living systems. The development of emissive optical probes is essential for improving our observation of intracellular signaling and recognition processes. Following excitation of the probe, photons emitted from the probe may be observed by spectroscopy or microscopy and encode information about their environments in their energy, lifetime, and polarization. Such optical probes may be based on organic fluorophores, quantum dots, recombinant proteins, or emissive metal complexes. In this Account, we trace the emergence of lanthanide coordination complexes as emissive optical probes. These probes benefit from sharp emission bands and long lifetimes. We can design these complexes to report on the concentration of key biochemical variables by modulation of spectral form, lifetime, or circular polarization. These properties allow us to apply ratiometric methods of analysis in spectroscopy or microscopy to report on local pH, pM (M = Ca, Zn), or the concentration of certain anionic metabolites, such as citrate, lactate, bicarbonate, or urate. For optical microscopy studies in living cells, these probes must be cell-permeable and, ideally, should localize in a given cell organelle. We undertook systematic studies of more than 60 emissive complexes, examining the time dependence of cellular uptake and compartmentalization, cellular toxicity, protein affinity, and quenching sensitivity. These results and their relationship to probe structure have allowed us to identify certain structure-activity relationships. The nature and linkage mode of the integral sensitizing group-introduced to harvest incident light efficiently-is of primary importance in determining protein affinity and cellular uptake and trafficking. In many cases, uptake may occur via macropinocytosis. We have defined three main classes of behavior: complexes exhibit predominant localization profiles in protein-rich regions (nucleoli/ribosomes), in cellular mitochondria, or in endosomes/lysosomes. Therefore, these systems offer considerable promise as intracellular optical probes, amenable to single- or two-photon excitation, that may report on the local ionic composition of living cells subjected to differing environmental stresses.
Accounts of Chemical Research 03/2009; 42(7):925-37. · 21.64 Impact Factor
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ABSTRACT: Five structurally related europium(iii) complexes of heptadentate macrocyclic ligands bearing azaxanthone or azathiaxanthone chromophores have been evaluated comparatively as responsive probes of the intracellular environment. Protein binding (HSA) and oxy-anion binding constants have been measured by titrimetric analysis, examining ratiometric emission changes for three examples, and systems exhibiting selectivity for citrate or hydrogencarbonate identified. The cytotoxicity of each ligand and complex has been assessed and correlated with the observed chemical stability profile in competitive aqueous media. The europium complex of L(2) is non-toxic, exhibits a large change in its emission spectral profile to variation in HCO(3)(-) (or citrate) concentration allowing ratiometric analysis, and localises in cellular mitochondria. Such features augur well for its future application as a responsive probe in microscopy to monitor local changes in pHCO(3).
Organic & Biomolecular Chemistry 07/2008; 6(12):2085-94. · 3.70 Impact Factor
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ABSTRACT: A set of three pH-responsive ratiometric Eu(III)complexes has been synthesised incorporating a coordinated azathioxanthone sensitiser and a pH dependent alkylsulfonamide moiety. Emission properties, anion binding affinities, pH response curves and protein binding constants were studied in detail in aqueous media, and solutions containing various concentrations of interfering anions and protein were also examined. The complex, [EuL3] exhibited some interference from protein and endogenous anions, e.g. lactate and hydrogen carbonate, but possessed a protonation constant of 7.2 in human serum solution. A suitable calibration curve was obtained and was used to determine the local pH using a 680/589 nm intensity ratio vs. pH plot. Confocal fluorescence microscopy images revealed fast uptake of the complex and a well distributed localisation within the cell; fast egress also occurred. Ribosomal localisation, with a high concentration within the protein-dense nucleoli was observed, in a similar manner to structurally related complexes bearing the same coordinated sensitising moiety. An IC(50) value of 67 (+/-20) microM was estimated using an MTT assay. Selected emission band ratio versus pH plots allow pH measurement in the range 6 to 8, enabling intracellular pH to be measured by microscopy. A value of 7.4 was estimated for NIH 3T3 cells in the protein rich regions of the nucleolus and ribosomes.
Organic & Biomolecular Chemistry 04/2008; 6(6):1020-33. · 3.70 Impact Factor
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ABSTRACT: Observation of two-photon excitation (760 nm) and emission of two responsive water soluble europium complexes is reported with cross-sections of up to 2 GM. Two-photon excitation spectra have also been measured, acquisition being achieved by the use of a cavity-dumped mode locked Ti-sapphire laser. Time-gated detection is used to differentiate the ligand fluorescence and metal centred emission in these europium complexes.
Dalton Transactions 01/2008; · 3.84 Impact Factor
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ABSTRACT: Following excitation in the range 370-405 nm, the emission spectrum of a cell permeable macrocyclic Eu(III) complex incorporating an N-methylsulfonamide moiety changes form with pH, allowing ratiometric pH measurements in the range 6 to 8.
Chemical Communications 03/2007; · 6.17 Impact Factor
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Paul Atkinson,
Karen S Findlay,
Filip Kielar, Robert Pal,
David Parker,
Robert A Poole,
Horst Puschmann,
Siobhan L Richardson,
Philip A Stenson,
Amber L Thompson,
Junhau Yu
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ABSTRACT: Several azaxanthone and azathioxanthone sensitising chromophores have been incorporated into macrocyclic ligands and form well-defined Eu and Tb complexes in polar media. Excitation of the heterocyclic chromophore in the range 330 to 382 nm leads to modest amounts of aromatic fluorescence and relatively efficient metal-based luminescence, with absolute metal-based quantum yields of up to 24% in aqueous media.
Organic & Biomolecular Chemistry 06/2006; 4(9):1707-22. · 3.70 Impact Factor
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ABSTRACT: A europium complex selectively staining the nucleolus of NIH 3T3, HeLa, and HDF cells is reported. This complex possesses not only the advantage of the long lifetime of europium emission (0.3 ms), but also a chromophore that allows excitation at a relatively long wavelength (lambda(max) = 384 nm) and gives rise to an acceptable quantum yield (9%). The complex can be used both in live cell and fixed cell imaging, giving an average intracellular concentration on the order of 0.5 microM. Strong binding to serum albumin has been demonstrated by examination of the analogous gadolinium complex, studying relaxivity changes with increasing protein concentration. The intracellular speciation of the complex has been examined by circularly polarized emission spectroscopy and is consistent with the presence of more than one europium species, possibly protein bound.
Journal of the American Chemical Society 03/2006; 128(7):2294-9. · 9.91 Impact Factor
