Rare earth cryptates for the investigation of molecular interactions in vitro and in living cells

Ghose, Sraboni; Trinquet, Eric; Laget, Michel; Bazin, Hervé; Mathis, Gérard

doi:10.1016/j.jallcom.2007.04.054

Cited by 28 publications

(16 citation statements)

References 13 publications

(17 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…46 At this point it should be noted that for a given terbium complex, its europium analogue will typically prove to be less sensitive to quenching with respect to processes operating by deactivation of the triplet excited state of the chromophore. This is because the energy of the 5 48 Meanwhile, direct quenching of this state has in some cases been attributed to an electron transfer process, 'driven' by the free energy it possesses. In the 2007 study outlined in the previous section, the quenching of europium and terbium luminescence by iodide was shown to occur via this mechanism.…”

Section: Quenching Of the Sensitiser Triplet Statementioning

confidence: 99%

Lanthanide complexes as chiral probes exploiting circularly polarized luminescence

2012

View full text Add to dashboard Cite

The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that:• a full bibliographic reference is made to the original source• a link is made to the metadata record in Durham E-Theses• the full-text is not changed in any wayThe full-text must not be sold in any format or medium without the formal permission of the copyright holders. Statement of CopyrightThe copyright of this thesis rests with the author. No quotations should be published without prior consent and information derived from it must be acknowledged. ABSTRACT -ii - ABSTRACTA series of studies has been undertaken to facilitate the identification and development of chiral lanthanide complexes that are able to report on changes in their local environment through modulation of the circular polarization of their emission. Reports of such systems remain relatively rare in the literature, notwithstanding the prevalence and importance of chirality in biological systems.The work described herein is separated into five chapters, the first of which comprises a discussion of relevant background information, along with a comprehensive review of responsive lanthanide-based CPL probes reported to date. A classification of these probes is built up, which informs the content of the following three chapters.Chapter 2 describes work undertaken in the pursuit of a novel lanthanide-based system for use as a CPL probe for the detection of proteins. The synthesis of an enantiopure lanthanide complex was undertaken and characterisation of this system carried out with reference to a structurally related racemic complex. A series of comparative investigations designed to probe the relative protein binding capability of these complexes was subsequently performed, which revealed that the observation of induced CPL from racemic lanthanide systems may be brought about by a change in complex constitution. This is the first example of such an effect from a well-defined racemic lanthanide complex in solution. Chapter 3 goes on to detail studies undertaken to demonstrate the utility of this racemic lanthanide system as a probe for chiral detection.Chapter 4 describes investigations carried out in an attempt to identify new systems exhibiting chiral quenching effects in solution. Initially, two pairs of enantiomeric electron-rich quenching species were assessed for their ability to quench the emission from an enantiopure DOTA-derived lanthanide complex differentially. Subsequently, investigations were focussed on examining the quenching of emission from novel enantiopure lanthanide complexes based on a 1,4,7-triazacyclononane framework, using cobalt complexes as the quenching species.Finally, Chapter 5 contains experimental procedures for each compound synthesised, as well as general experimental procedures.

show abstract

Section: Quenching Of the Sensitiser Triplet Statementioning

confidence: 99%

Lanthanide complexes as chiral probes exploiting circularly polarized luminescence

2012

View full text Add to dashboard Cite

show abstract

“…(5–9) TRL detection of resonance energy transfer between LC donors and short-lifetime organic or protein fluorophore acceptors has been exploited extensively for high-throughput, cell-free assays of biomolecular interactions, and picomolar detection limits of LC-labeled analytes have been achieved. (4,10–13) Other long-lived probes have also been developed for TRL applications, including metal-ligand complexes of platinum and palladium that emit with shorter lifetimes than LCs (100 ns – 10 μs) and that can be excited with visible light. (14–16)…”

Section: Introductionmentioning

confidence: 99%

Time‐resolved microscopy for imaging lanthanide luminescence in living cells

2010

View full text Add to dashboard Cite

Time-resolved luminescence (TRL) microscopy can image signals from lanthanide coordination complexes or other probes with long emission lifetimes, thereby eliminating short-lifetime (\100 ns) autofluorescence background from biological specimens. However, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes, making it difficult to rapidly acquire high quality images at probe concentrations that are relevant to live cell experiments. This article describes the development and characterization of a TRL microscope that employs a light-emitting diode (LED, k em 5 365 nm) for pulsed epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection. Europium chelate-impregnated microspheres were used to evaluate instrument performance in terms of short-lifetime fluorescence background rejection, photon collection efficiency, image contrast, and signal-to-noise ratio (SNR). About 200 nm microspheres were imaged within the time resolution limit of the ICCD (66.7 ms) with complete autofluorescence suppression. About 40 nm microspheres containing $400 chelate molecules were detected within $1-s acquisition times. A luminescent terbium complex, Lumi4-Tb 1 , was introduced into the cytoplasm of cultured cells at an estimated concentration of 300 nM by the method of osmotic lysis of pinocytic vesicles. Time-resolved images of the living, terbium complex-loaded cells were acquired within acquisition times as short as 333 ms, and the effects of increased exposure time and frame summing on image contrast and SNR were evaluated. The performance analyses show that TRL microscopy is sufficiently sensitive and precise to allow high-resolution, quantitative imaging of lanthanide luminescence in living cells under physiologically relevant experimental conditions. ' 2010 International Society for Advancement of Cytometry Key terms fluorescence microscopy; lanthanide; time-resolved; signal-to-noise ratio THE sensitivity and precision of fluorescence-based bioassays and microscopy is often diminished by autofluorescence background emitted from samples and containers or coverslips. Time-resolved luminescence (TRL) measurements can effectively eliminate autofluorescence when the detected analyte has an emission lifetime exceeding that of endogenous fluorophores (\100 ns) (1). TRL instrumentation uses a finite pulse of light to excite a sample (Fig. 1). Then, the detector is electronically switched on, or unshuttered after a short interval (the gate delay) during which sample autofluorescence has diminished. The detector remains on for a finite interval (the gate width), and the output from multiple excitation/emission cycles can be integrated to increase signal.The most common emissive probes for TRL-based bioassays are ligand-sensitized, coordination complexes of lanthanide cations. Lanthanide complexes (LCs) have long lifetimes (ls-ms) that facilitate TRL detection as well as large Stokes shifts ([150 nm) and multiple, narrow emission bands that make ...

show abstract

“…The ability to temporally and spectrally isolate lanthanide emission signals makes it possible to detect analytes at small concentrations (pM – nM) in complex matrices, and lanthanide-based assays are routinely used for diagnostics and high throughput screening using commercial plate reader instrumentation. 3–4 In recent years, there has been considerable interest in leveraging the inherent sensitivity of TGD with Tb(III) and Eu(III) complexes for applications in live-cell microscopic imaging. 1–2 In this context, the chemistry and photophysics of lanthanide complexes must be considered in relation to the workings and limitations of microscopes, the interaction and compatibility of complexes with cells, and the nature of the biological questions that are typically addressed by live-cell imaging experiments.…”

Section: Introductionmentioning

confidence: 99%

Lanthanide-Based Imaging of Protein–Protein Interactions in Live Cells

2013

View full text Add to dashboard Cite

In order to deduce the molecular mechanisms of biological function, it is necessary to monitor changes in the sub-cellular location, activation and interaction of proteins within living cells in real time. Förster resonance energy transfer (FRET)-based biosensors that incorporate genetically encoded, fluorescent proteins permit high spatial resolution imaging of protein–protein interactions or protein conformational dynamics. However, non-specific fluorescence background often obscures small FRET signal changes, and intensity-based biosensor measurements require careful interpretation and several control experiments. These problems can be overcome by using lanthanide (Tb(III) or Eu(III)) complexes as donors and green fluorescent protein (GFP) or other conventional fluorophores as acceptors. Essential features of this approach are the long-lifetime (~ms) luminescence of Tb(III) complexes and time-gated luminescence microscopy. This allows pulsed excitation followed by a brief delay that eliminates nonspecific fluorescence before detection of Tb(III)-to-GFP emission. The challenges of intracellular delivery, selective protein labeling, and time-gated imaging of lanthanide luminescence are presented, and recent efforts to investigate the cellular uptake of lanthanide probes are reviewed. Data is presented showing that conjugation to arginine-rich, cell penetrating peptides (CPPs) can be used as a general strategy for cellular delivery of membrane impermeable lanthanide complexes. A heterodimer of a luminescent Tb(III) complex, Lumi4, linked to trimethoprim (TMP) and conjugated to nonaarginine via a reducible disulfide linker rapidly (~10 min) translocates into the cytoplasm of Maden Darby canine kidney cells from culture medium. With this reagent, the intracellular interaction between GFP fused to FK506 binding protein 12 (GFP-FKBP12) and the rapamycin binding domain of mTOR fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) was imaged at high signal-to-noise ratio with fast (1–3 s) image acquisition using a time-gated luminescence microscope. The data reviewed and presented here show that lanthanide biosensors enable fast, sensitive and technically simple imaging of protein-protein interactions in live cells.

show abstract

Rare earth cryptates for the investigation of molecular interactions in vitro and in living cells

Cited by 28 publications

References 13 publications

Lanthanide complexes as chiral probes exploiting circularly polarized luminescence

Lanthanide complexes as chiral probes exploiting circularly polarized luminescence

Time‐resolved microscopy for imaging lanthanide luminescence in living cells

Lanthanide-Based Imaging of Protein–Protein Interactions in Live Cells

Contact Info

Product

Resources

About