Lanthanide Emission Research Articles

Time Gating Improves Sensitivity in Energy Transfer Assays with Terbium Chelate/Dark Quencher Oligonucleotide Probes

Lanthanides are attractive as biolabels because their long luminescence decay rates allow time-gated detection, which separates background scattering and fluorescence from the lanthanide emission. A stable and highly luminescent terbium complex based on a tetraisophthalamide (TIAM) chelate is paired with a polyaromatic-azo dark quencher (referred to as a Black Hole Quencher or BHQ) to prepare a series of 5'TIAM(Tb)/3'BHQ dual-labeled oligonucleotide probes with no secondary structure. Luminescence quenching efficiency within terbium/BHQ probes is very dependent on the terbium-BHQ distance. In an intact probe, the average terbium-BHQ distance is short, and Tb --> BHQ energy transfer is efficient, decreasing both the terbium emission intensity and lifetime. Upon hybridization or nuclease digestion, which spatially separate the Tb and BHQ moieties, the Tb luminescence intensity and lifetime increase. As a result, time-gated detection increases the emission intensity ratio of the unquenched probe/quenched probe due to the shorter lifetime of the quenched species. A 40-mer probe that has a 3-fold increase in steady-state luminescence upon digestion has a 50-fold increase when gated detection is used. This study demonstrates that time gating with lanthanide/dark quencher probes in energy transfer assays is an effective means of improving sensitivity.

Green upconversion fluorescence in terbium coordination complexes

Three terbium coordination complexes have been synthesized. Bright green emission from these compounds can be obtained under infrared pulsed laser pumping. The detail photophysics demonstrates it is due to two-photon absorption of the ligand. In addition, the effect of different ligands on upconversion efficiency was compared and discussed. This study demonstrates that enhanced two-photon induced lanthanide emission can be achieved when it is sensitized with suitable organic ligands. The estimated upconversion efficiency was 1% with an excitation energy of 1.2 W/cm 2 under nanosecond pulse excitation, suggesting their potential applications in two-photon area.

Novel Ligands Incorporating Phosphinoxide Groups as Particularly Efficient Sensitizers for Lanthanide Emission

Several novel phosphinoxide ligands have been obtained, based on molecular scaffolds: pyridine, 2,2′-bipyridine, cavitands, cyclotriphosphazene and 1,3,5-triazine. These compounds form highly luminescent complexes with Eu(III) and Tb(III). Particularly good luminescent properties were observed in case of cyclotriphosphazene, pyridine and 2,2′-bipyridine ligands with photoluminescence quantum yields approaching 25%. Cavitand-based phosphinoxides turned out to be rather poor sensitizers for the lanthanide ion emission. Particularly long emission lifetime was observed in case of pyridine P-oxide complexes of Eu(III) and Tb(III), exceeding 3 ms.

Photoluminescence investigation of europium-doped alumina, titania and indium sol–gel-derived films in porous anodic alumina

Photoluminescence (PL) properties of Eu-doped xerogel films fabricated on porous anodic alumina (PAA) and flat substrates are analysed with respect to the xerogel content (TiO 2, Al 2O 3, In 2O 3) and excitation wavelength. A strong enhancement of Eu-related emission in the broad UV range of the excitation has been found for the three xerogel systems. However, the enhancement factor is not simple to interpret, it possesses rather a complex nature. We have proposed that the increase of PL intensity is composed of two components. The first component is associated with an improvement of the energy transfer from the host xerogel matrix to lanthanide ions. The second one is evoked by multiple scattering of exciting light in the pore channels. This additional path of the excitation and energy transfer improves essentially the PL of lanthanide ions. Moreover, in periodic matrix of PAA the emission of lanthanide is redistributed by angle contributing into PL enhancement.

Unexpected Sensitization Efficiency of the Near-Infrared Nd3+, Er3+, and Yb3+Emission by Fluorescein Compared to Eosin and Erythrosin

Near-infrared emissive lanthanide complexes were synthesized with covalently attached sensitizers that absorb in the visible. This functionalization was designed such that the sensitizer is in close proximity to the lanthanide ion, which is a prerequisite for efficient energy transfer from the excited sensitizer to the lanthanide ion. The sensitizers used were fluorescein, eosin, and erythrosin, which were linked via a -alanine spacer to the polydentate chelate. The sensitizers were chosen because they absorb visible light and are structurally very similar, but the intrinsic intersystem crossing quantum yields of the sensitizers vary significantly, because of the presence of the heavy atoms (bromine in eosin and iodine in erythrosin). It was expected that an intrinsic high intersystem crossing would be beneficial in the sensitization process, because energy transfer occurs through the triplet state of sensitizers. However, because of the enhanced intersystem crossing of the sensitizers by the nearby heavy and paramagnetic lanthanide ions, these intrinsic differences were largely diminished. It was even found that fluorescein acts as a more efficient sensitizer for the NIR emission than eosin and erythrosin. The donating triplet state of fluorescein is higher in energy than that of eosin and erythrosin, resulting in less energy back transfer and therefore in a higher efficiency of sensitized emission. This and considerations of selection rules for energy transfer to the lanthanide ions made it possible to distinguish the 4F9/2 level of Nd3+ as the main acceptor channel for energy transfer. In the Er3+ complexes, the enhancement in intersystem crossing was lower in the eosin and erythrosin complexes than in the fluorescein complex, which was concluded from the remaining complex fluorescence. Furthermore, it is tentatively concluded that additional pathways other than those allowed in Dexter energy transfer play a role in the sensitization of Er3+. In the Yb3+ complexes, the higher efficiency of sensitization by fluorescein is due to the enhanced intersystem crossing that is larger in the fluorescein complex than in the eosin or erythrosin complex.

Crown ether lanthanide complexes as building blocks for luminescent ternary complexes

Lanthanide complexes of the macrocyclic ligands 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane- N, N′-diacetic acid (H 2(dacda)) and 1,4,7,10-tetraoxa-13-azacyclopentadecane-13-acetic acid (H(macma)) have been isolated and characterised. The photophysical properties of the red and green emissive, Eu(III) and Tb(III), respectively, complexes have been elucidated. Upon addition of mono- or bi-dentate aromatic carboxylates or dibenzoylmethide in solutions of the Eu(III) macrocycles triggering of the lanthanide emission is observed. The formation of the ternary complex between the lanthanide macrocycle and the sensitiser is controlled by the available coordinaton sites of the lanthanide macrocycle prior to the binding event.

Studying Sigma–Core Interactions in Escherichia coli RNA Polymerase by Electrophoretic Shift Assays and Luminescence Resonance Energy Transfer

Publisher Summary This chapter aims to describe electrophoretic mobility shift (EMS) assays and fluorescence resonance energy transfer (FRET) for several reasons. EMS assays are fairly simple and quick to perform with the equipment present in most biologically oriented laboratories. At the same time, they give useful initial information about a protein binding to another protein, DNA, or RNA. EMS assays are based on the change of mobility of a protein during polyacrylamide gel electrophoresis (PAGE) on binding to DNA, RNA, or another protein. Crucial to EMS assays the fact that the procedure involves separating the complex from the unbound binding partner by size and charge differences is discussed. This changes the equilibrium conditions at which initial binding occurs, and thus weak interactions, such as in complexes that have a half-life shorter than the time scale of the separation step, are under-represented, or cannot be detected, as the interaction does not persist throughout the procedure. It focuses on luminescence resonance energy transfer (LRET)-based assays for a homogeneous assay to measure formation of the σ70– β complex. LRET is a recent modification of FRET that uses a lanthanide-based donor fluorophore. The more general term luminescence instead of fluorescence indicates that lanthanide emission is technically not fluorescence.

Accumulation and elimination of lanthanum by duckweed (Lemna minor L.) As influenced by organism growth and lanthanum sorption to glass

Lanthanide emissions to the environment increase as a result of the growing industrial applications of these elements. However, robust data to evaluate the environmental fate of lanthanides are scarce. This article describes the accumulation and elimination of lanthanum (La) by common duckweed (Lemna minor L.). Speciation modeling was performed to assure that solubility products were not exceeded. It also showed that La was predominantly associated with ethylenediaminetetraacetic acid (EDTA). Lanthanum concentrations in plants and medium and the amounts sorbed to glass vessels were quantified by using the radioisotope 140La. The amount of La adsorbed on the glass reached values of 25% of the total La present. A model was formulated to describe La uptake in exponentially growing duckweed in the presence of an adsorptive surface. Growth-induced dilution appeared more efficient in lowering plant La concentrations than actual elimination. An elimination study revealed two compartments, of which the smallest eliminated 50 times faster than the bigger compartment, which eliminated mainly by growth dilution. The average bioconcentration factor was 2,000 L/kg fresh weight and 30,000 L/kg dry weight, comparable with those of other higher plants. At the applied concentration of 10 nM, no effects were observed on duckweed growth. However, the high bioconcentration factor warrants monitoring of lanthanide emissions.

Pressure-induced dark-to-bright transition inLu2O3:Ce3+

Application of pressure to the nonemitting system ${\mathrm{Lu}}_{2}{\mathrm{O}}_{3}:{\mathrm{Ce}}^{3+}$ leads to the generation of luminescence in the visible. The luminescence is assigned to the $5\stackrel{\ensuremath{\rightarrow}}{d}4f$ transition of ${\mathrm{Ce}}^{3+},$ and is attributed to a pressure-induced electronic crossover of the excited $5d$ state of ${\mathrm{Ce}}^{3+}$ and the conduction-band edge of ${\mathrm{Lu}}_{2}{\mathrm{O}}_{3}.$ The results yield insight into the emission efficiency of $5\stackrel{\ensuremath{\rightarrow}}{d}4f$ transitions, and provide a foundation for advancing new phosphors and tunable laser materials in the visible and ultraviolet based on the broad $5\stackrel{\ensuremath{\rightarrow}}{d}4f$ emission of lanthanides.

Sensitization of lanthanides by nonnatural amino acids.

The sensitization of Eu(III) and Tb(III) by ethylenediaminetetraaceticacid (EDTA)-derivatized tryptophan (Trp), 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) has been examined. These Trp analogs were utilized in the present study because they can be incorporated into proteins in place of native Trp residues and because they absorb strongly beyond 305 nm (where Trp absorbance goes to zero), allowing selective excitation of such species in the presence of other Trp-containing proteins. All three indole derivatives were able to sensitize Tb(III) luminescence, with the relative sensitization being in the order Trp > 5HW > 7AW. On the other hand, only the 7AW-EDTA complex was able to sensitize Eu(III) luminescence, likely owing to a better spectral overlap between 7AW emission and Eu(III) absorbance. The sensitized emission of Tb(III) and Eu(II) displayed the expected long emission lifetimes at 545 nm [for Tb(III)] and 617 nm [for Eu(III)], indicating that long-lifetime lanthanide emission could be produced using nonnatural amino-acid donors. Thus, 7AW- and 5HW-sensitized lanthanide emissions should prove to be useful in biophysical studies, such as the use of fluorescence energy transfer to probe biomolecular interactions in vivo.

Survey of factors determining the circularly polarised luminescence of macrocyclic lanthanide complexes in solution.

The development of emissive lanthanide complexes as structural or reactive probes to signal changes in their local chiral or ionic environment has been inhibited by the lack of understanding of correlating structural and electronic spectral information. The definition of relatively rigid enantiopure macrocyclic lanthanide complexes, whose inter- and intramolecular exchange dynamics have been defined, offers scope for remedying this situation. Chiral axially symmetric lanthanide complexes in solution give rise to large emission dissymmetry values (g(em)) in CPL spectra. The sign and magnitude of g(em) are determined by the degree of twist about the principal axis, which is predicted to be a maximum at +/-22.5 degrees, and by the site symmetry and local ligand field. In particular, the polarisability of the ligand donor atoms, especially for any axial donor, is very important. Examples of each case are discussed for structurally related cationic Eu(III) complexes.

Synthesis and photochemical properties of new coumarin-derived ionophores and their alkaline-earth and lanthanide complexes

Several new ionophores derived from crown ethers and iminodiacetic subunits attached to 3-aroylcoumarins have been synthesized and fully characterized. The alkaline-earth complexes of these new ligands were studied from their UV–visible and fluorescence data. Some systems displayed strong bathochromic shifts upon complexation with Mg 2+ that may make them useful signaling devices of this cation. The corresponding Eu 3+ and Tb 3+ chelates were easily formed and their photophysical properties were measured. In all the cases, lanthanide emission lifetimes were in the range of milliseconds albeit quantum yields were low. Possible energy-transfer mechanisms are discussed.

A novel Eu(III)-based luminescent chemosensor: determining pH in a highly acidic environment

The design, synthesis and photophysical evaluation of the Eu(III)-based chemosensor [Eu·1], is described. The sensors show large (ca. 300 fold) pH dependent luminescence enhancements for the Eu(III) lanthanide emission, i.e. it is ‘switched on’ in highly acidic media, between pH ca. 1.5 and 3.5 with a pKa of 2.4 (±0.1) when excited at 350 nm. The fluorescence spectra of [Eu·1], consisting of the quinoline emission, is also pH dependent, where the fluorescence is ‘switched on’ at 458 nm, in the pH region of 3–6, but the emission is quenched between pH 1.5 and 3. Both pH-intensity profiles display an ‘off–on–off’ bell-shape behaviour similar to that seen for many enzymatic pH dependent processes.

Luminescence Resonance Energy Transfer Analysis of RNA Polymerase Complexes

Resonance energy transfer allows measurement of atomic-scale distances under a variety of solution conditions. Luminescence resonance energy transfer (LRET) is a variant of energy transfer measurement in which lanthanide chelates are used as the probes. The unusual properties of lanthanide emission, in particular their long microsecond-scale lifetimes, offer several advantages for energy transfer measurements with biological samples. One of the unique features of LRET is the ability to measure energy transfer under conditions where severe heterogeneity of labeled macromolecules exists. This feature of LRET is the special emphasis of this article. We describe here LRET methodology with a particular attention to using sensitized acceptor emission to determine efficiency of energy transfer. Although we employed this technique in the characterization of Escherichia coli RNA polymerase complexes it is readily compatible with the study of essentially any protein.

Quantum yields of luminescent lanthanide chelates and far-red dyes measured by resonance energy transfer.

Luminescent lanthanide chelates have unusual spectroscopic characteristics that make them valuable alternative probes to conventional organic fluorophores. However, fundamental parameters such as their quantum yield, and radiative and nonradiative decay rates have been difficult or impossible to measure. We have developed a simple and robust method based on resonance energy transfer to accurately measure these parameters. In addition, the excitation/emission process in lanthanide chelates involves several steps, and we are able to quantify each step. These include excitation of an organic antenna, transfer of energy from the antenna to lanthanide, and then lanthanide emission. Overall, the parameters show that lanthanide chelates can be efficient long-lived emitters, making them sensitive detection reagents and excellent donors in resonance energy transfer. The method is also shown to be applicable to photophysical characterization of red-emitting dyes, which are difficult to characterize by conventional means.

Novel polyaminocarboxylate chelates derived from 3-aroylcoumarins

We have devised efficient reaction pathways to attach aminopolycarboxylate subunits to the 3-aroylcoumarin chromophore. Two series of compounds were thus prepared in which the chelating arms were directly bonded to the coumarin ring (series A) or to the 3-aroyl moiety (series B). The corresponding Eu(III) and Tb(III) chelates were easily formed and their photophysical properties measured. In all the cases, lanthanide emission lifetimes were in the range of ms. Unfortunately, quantum yields were relatively low. Measurement of T1 states gave too low range of values to sensitize Tb(III). In fact, the metal emission of Tb(III) chelates of series A was not observed. However, series B was able to sensitize both metals. The absorption/energy-transfer/emission mechanisms are discussed.

An improved instrument for measuring time-resolved lanthanide emission and resonance energy transfer

Time-resolved fluorescence, particularly of millisecond lanthanide emission, is of considerable interest because of potentially increased sensitivity when short-lived autofluorescence is a problem. Lanthanides have also previously been shown to be excellent donors in resonance energy transfer measurements, where a lanthanide donor transfers energy to a conventional fluorophore. An instrument for the detection of micro- to millisecond lanthanide luminescence and delayed fluorescence, amidst a background of large prompt fluorescence, is described. Time-delayed emission spectra can be measured with 16 μs dead time while suppressing prompt fluorescence by 25 000-fold. This is accomplished by using a mechanical chopper placed very close to the entrance slit of a diffraction spectrometer, and a charge coupled device detector at the exit port. Excited-state lifetimes can also be measured with a 22 μs dead time while reducing detector ringing due to prompt fluorescence to an insignificant level. This is accomplished by high-voltage gating of a photon-counting photomultiplier tube in combination with a fast constant-fraction discriminator and amplifier. These improvements, which utilize all commercially available equipment, reduce the dead time by a factor of 10 for delayed emission measurements and a factor of 5 for lifetime measurements over a previously described instrument. The current instrument is particularly well suited for lanthanide-based resonance energy transfer measurements.

Reversible anion binding in aqueous solution at a cationic heptacoordinate lanthanide centre: selective bicarbonate sensing by time-delayed luminescence

Reversible displacement of up to two metal bound water molecules at a coordinately unsaturated, cationic lanthanide centre is signalled by increases in the luminescence intensity or lifetime of the emissive lanthanide: selective chelation of hydrogencarbonate is consistent with emission spectroscopic, ESMS and CPL measurements.

Kinetic determination of bromadiolone based on lanthanide-sensitized luminescence

A very fast, selective kinetic method for the determination of bromadiolone is proposed. The analytical parameter used is the initial rate of the luminescence reaction between bromadiolone and terbium(III) in the presence of Triton X-100. The luminescence signal arises from an intramolecular energy transfer process between excited bromadiolone and terbium(III), which is followed by the lanthanide emission. The use of stopped-flow mixing technique allows to obtain kinetic data within only 0.2 s and automatize the method. The calibration graph is linear over the range 1.5–18 μg ml −1 bromadiolone and the detection limit is 0.3 μg ml −1. The mid-range relative standard deviation is close to 2%. The recoveries obtained by applying the method directly to the analysis of irrigation water samples ranged from 86.7 to 100.0%.

Water-Soluble Neutral Calix[4]arene-Lanthanide Complexes: Synthesis and Luminescence Properties.

Water-soluble calix[4]arenes 10a,b with chromophores ("antenna") attached to the lower rim via a short spacer are described. In the neutral lanthanide complexes of 10a,b photoexcitation of the antenna induces lanthanide emission via intramolecular energy transfer. Calix[4]arene 10b with a chrysene moiety as sensitizer shows strong lanthanide emission for Eu(3+) with an excitation maximum at lambda = 363 nm.