Increasing lanthanide luminescence by use of the RETEL effect.

BACKGROUND: Luminescent lanthanide complexes produce emissions with the narrowest-known width at half maximum; however, their significant use in cytometry required an increase in luminescence intensity. The companion review, Leif et al., Cytometry 2006;69A:767-778, described a new technique for the enhancement of lanthanide luminescence, the Resonance Energy Transfer Enhanced Luminescence (RETEL) effect, which increases luminescence and is compatible with standard slide microscopy. METHODS: The luminescence of the europium ion macrocyclic complex, EuMac, was increased by employing the RETEL effect. After adding the nonluminescent gadolinium ion complex of the thenoyltrifluoroacetonate (TTFA) ligand or the sodium salt of TTFA in ethanol solution, the EuMac-labeled sample was allowed to dry. Both a conventional arc lamp and a time-gated UV LED served as light sources for microscopic imaging. The emission intensity was measured with a CCD camera. Multiple time-gated images were summed with special software to permit analysis and effective presentation of the final image. RESULTS: With the RETEL effect, the luminescence of the EuMac-streptavidin conjugate increased at least six-fold upon drying. Nuclei of apoptotic cells were stained with DAPI and tailed with 5BrdUrd to which a EuMac-anti-5BrdU conjugate was subsequently attached. Time-gated images showed the long-lived EuMac luminescence but did not show the short-lived DAPI fluorescence. Imaging of DNA-synthesizing cells with an arc lamp showed that both S phase and apoptotic cells were labeled, and that their labeling patterns were different. The images of the luminescent EuMac and fluorescent DAPI were combined to produce a color image on a white background. This combination of simple chemistry, instrumentation, and presentation should make possible the inexpensive use of the lanthanide macrocycles, Quantum Dyes, as molecular diagnostics for cytological and histopathological microscopic imaging.     

Action cross sections of two-photon excited luminescence of some Eu(III) and Tb(III) complexes.

Four different luminescent lanthanide complexes have been studied with respect to multiphoton excitation using near-infrared femtosecond pulses. The method for measuring action cross sections of two-photon excited fluorescence in solution relative to a known standard is reviewed. Two refractive index-related corrections are necessary in this method: one for the multiphoton excitation process, the other for the collection of the emitted light. It has been found that (2,4,6-trimethoxyphenyl)dipicolinic acid and Michler's ketone are reasonable sensitisers of two-photon excited lanthanide luminescence in solution, whereas dipicolinic acid and carbostyril-124 do not give rise to any detectable two-photon excited lanthanide luminescence using modest excitation powers (<20 mW focused at the sample) in the 700-1000 nm range.     

Synthesis and luminescence properties of lanthanide complexes with a new tripodal ligand featuring N‐thenylsalicylamide arms

To explore the relationship between the structure of the ligands and the luminescent properties of the lanthanide complexes, luminescent lanthanide complexes of a new tripodal ligand, featuring N‐thenylsalicylamide arms, were synthesized and characterized by elemental analysis, IR and TGA measurements. Photophysical properties of the complexes were studied by means of UV ? visible absorption and steady‐state luminescence spectroscopy. The results of UV ? vis spectra indicate that metal binding does not disturb the electronic structure of the ligand. Excited‐state luminescence lifetimes and quantum yields of the complexes were determined. The photoluminescence analysis suggested that there is an efficient ligand ? Ln(III) energy transfer for the Tb(III) complex, and the ligand is an efficient 'antenna' for Tb(III). From a more general perspective, the results demonstrated the potential application of the lanthanide complex as luminescent materials in material chemistry. Copyright © 2012 John Wiley & Sons, Ltd.     

Analogs of Eu3+ DOTAM-Gly-Phe-OH and Tm3+ DOTAM-Gly-Lys-OH: synthesis and magnetic properties of potential PARACEST MRI contrast agents

Chelated lanthanide ions, especially gadolinium, have found wide use as contrast agents in magnetic resonance imaging. A new paradigm for generating contrast, termed PARACEST, was recently described that requires the slow exchange of water or other exchangeable protons present in the ligand framework. In previous work, we have described a synthetic method for the preparation of dipeptide conjugates of DOTAM for use as PARACEST agents. Two compounds possessed interesting magnetic properties: the Eu(3+) complex of DOTAM-Gly-Phe-OH and the Tm(3+) complex of DOTAM-Gly-Lys-OH. To understand the relationship between the structure of these complexes and their magnetic properties, we have expanded our synthetic methodology and prepared several new complexes. Ligands have been prepared in which the terminal phenylalanine moieties have been replaced with tryptophan or tyrosine, the distance to the amino acid residue possessing an alpha-substituent has been changed, or phenylalanine and lysine have been combined in the peptide sequence. The preparation of lanthanide(III) complexes of these ligands has been achieved and their PARACEST properties have been determined.     

Lanthanide binding and IgG affinity construct: Potential applications in solution NMR, MRI, and luminescence microscopy

Paramagnetic lanthanide ions when bound to proteins offer great potential for structural investigations that utilize solution nuclear magnetic resonance spectroscopy, magnetic resonance imaging, or optical microscopy. However, many proteins do not have native metal ion binding sites and engineering a chimeric protein to bind an ion while retaining affinity for a protein of interest represents a significant challenge. Here we report the characterization of an immunoglobulin G-binding protein redesigned to include a lanthanide binding motif in place of a loop between two helices (Z-L2LBT). It was shown to bind Tb(3+) with 130 nM affinity. Ions such as Dy(3+) , Yb(3+) , and Ce(3+) produce paramagnetic effects on NMR spectra and the utility of these effects is illustrated by their use in determining a structural model of the metal-complexed Z-L2LBT protein and a preliminary characterization of the dynamic distribution of IgG Fc glycan positions. Furthermore, this designed protein is demonstrated to be a novel IgG-binding reagent for magnetic resonance imaging (Z-L2LBT:Gd(3+) complex) and luminescence microscopy (Z-L2LBT: Tb(3+) complex).     

Analysis of collagen cyanogen bromide peptides using electrophoresis in continuous concave gradient polyacrylamide gels.

A rapid and simple spectrophotometric method is described for the estimation of microgram quantities of glycosaminoglycans following the formation of soluble complexes with alcian blue dye. The method is based on the different absorption spectra of the dye and dye-glycosaminoglycan complexes. No heating, centrifugation, lengthy equilibration, or sophisticated instrumentation which hamper other methods are required. Samples are mixed with freshly prepared dye solution and absorbance readings at 480 nm are compared to an appropriate standard curve. Albumin and individual monosaccharides do not interfere with the assay but high concentrations of chloride ion do. The method is suitable for the estimation of total glycosaminoglycan levels in biological fluids such as urine and blood.     

The thermodynamics of lanthanide ion binding to adriamycin

We have examined the thermodynamics of lanthanide ion binding to adriamycin by monitoring the effects of variations in temperature on the dissociation constants of various lanthanide ion complexes of the drug. These constants were obtained by analyzing the extent of quenching of the fluorecence of adriamycin in the presence of lanthanide ions in terms of an equilibrium binding process. Our binding model included the following features, all of which are supported by evidence derived from previous published reports, vide infra. The lanthanides form 1:1 complexes with adriamycin. The binding is dependent on the pH of the solution, indicating that only the nonprotonated amine form of the drug participates in lanthanide ion binding. And finally the drug self-associates in solution to for a dimeric species. Our present results indicate that the binding process is almost completely independent of temperature, indicating that the enthalpy of complex formation is extremely small. The entropy terms are consistent with the formation of a complex in which the adriamycin acts as a bidentate ligand. Our results suggest that the lanthanide complexes are isostructural, at least as far as the adriamycin is concerned, throughout the lanthanide series.     

Synthesis, structural characterization, and photo and electroluminescence of a novel terbium(III) complex: {Tris(acetylacetonate) [1,2,5]thiadiazolo[3,4-f][1,10]phenanthroline}terbium(III)

Lanthanides ion complexes have been intensively investigated as light emitting materials due to their interesting photophysical properties, such as narrow line luminescence with a long lifetime, large Stokes shift and high luminescence quantum efficiency. Here we report the synthesis, structural and photophysical properties of a new Tb(III) complex. This complex showed strong photoemission of green light both in solution and in the solid state as well as the characteristic emission lines of the Tb(III) ion. The electroluminescence properties of the complex were also studied and we obtained bright green light emission through the use of a co-deposited structure. The fabricated device showed a typical diode behavior with a low threshold bias voltage (around 10 V).     

αvβ3-Integrin-targeting lanthanide complex: synthesis and evaluation as a tumor-homing luminescent probe

The application of lanthanide complexes in the time-resolved fluorescence imaging of living cells has emerged in the last few decades, providing high-contrast images of cells through detection of the delayed emission. In the present study, we synthesized novel trivalent lanthanide complexes containing the cyclic peptide c(RGDfK) to visualize the α_vβ₃-integrin-expressing tumor cells. Conjugation of c(RGDfK) with the macrocyclic bipyridine ligand had little effect on the fluorescence properties of the complex, indicating that the coordinated lanthanide ion was well isolated from the peptide. Bright luminescence images of α_vβ₃-integrin-expressing U87-MG cells were successfully obtained by employing the probes.     

Lanthanide Circularly Polarized Luminescence: Bases and Applications

Lanthanide (III) luminescence is very characteristic: it is characterized by narrow emission bands, large Stokes shift, and a long excited state lifetime. Moreover, chiral lanthanide complexes can emit strongly circularly polarized light in a way that is almost precluded to purely organic molecules. Thanks to the sensitivity and specificity of the Ln circularly polarized luminescence (CPL) signal, CPL‐active complexes are therefore employed as bioanalytical tools and other uses can be envisaged in many other fields. Here we present a brief overview of the most recently developed CPL‐active lanthanide complexes and a selected few examples of their applications. We briefly discuss the main mechanisms that can rationalize the observed outstanding CPL properties of these systems, and some practical suggestions on how to measure and report data. Chirality 27:1–13, 2015. © 2014 Wiley Periodicals, Inc.     

Synthesis and luminescence properties of lanthanide complexes with a new tripodal ligands featuring salicylamide arms

A series of luminescent lanthanide complexes with a new tripodal ligand featuring salicylamide arms, 2,2′,2″‐nitrilotris(2‐furfurylaminoformylphenoxy)triethylamine (L), were synthesized and characterized by elemental analysis, IR and molar conductivity measurements. Photophysical properties of the complexes were studied by means of UV–vis absorption and steady‐state luminescence spectroscopy. Excited‐state luminescence lifetimes and quantum yield of the complexes were determined. Luminescence studies demonstrated that the tripodal ligand featuring salicylamide arms exhibits a good antennae effect with respect to the Tb(III) and Dy(III) ion due to efficient intersystem crossing and ligand to metal energy transfer. From a more general perspective, this work offers interesting perspectives for the development of efficient luminescent stains and enlarges the arsenal for developing novel luminescent lanthanide complexes of salicylamide derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

UV light‐tunable fluorescent inks and polymer hydrogel films based on carbon nanodots and lanthanide for enhancing anti‐counterfeiting

Novel water‐soluble green fluorescent carbon nanodots (CNs) using methacrylic acid and m‐phenylenediamine as precursors were first synthesized using a one‐pot hydrothermal method. Red fluorescent lanthanide complexes were prepared using lanthanide ion Eu³⁺ and pyridine‐2,6‐dicarboxylic acid. The optical properties of CNs were characterized using ultraviolet visible (UV) spectra and fluorescence spectra, microscopic morphology was characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the elemental composition was characterized using Fourier transform‐infrared spectroscopy (FT‐IR) and X‐ray photoelectron spectra (XPS). The fluorescence spectra of the lanthanide complexes were also measured. A simple strategy was developed to prepare UV light‐tunable fluorescent inks and polymer hydrogels films based on CNs and lanthanide complexes. The fluorescent inks and polymer hydrogels films could be repeatedly switched between green and red fluorescence. The change of color depended on luminescence of the CNs and the lanthanide complexes under 254 and 365 nm UV light, respectively. The UV light‐tunable fluorescent inks and polymer hydrogels films could enhance its anti‐counterfeiting function for data and information.     

Interactions between metal ions and carbohydrates. Syntheses and spectroscopic studies of several lanthanide nitrate-D-galactitol complexes

Two complexes of neutral D-galactitol (C(6)H(14)O(6), G) with terbium nitrate, TbGN(I) and TbGN(II), and one complex with samarium nitrate SmGN were synthesized and characterized. From IR, FIR, THz and luminescence spectra the possible coordinations were suggested, and the single-crystal X-ray diffraction results confirm the spectroscopic conclusions. In TbGN(I) (Tb(NO(3))(3)·C(6)H(14)O(6)·3H(2)O), the Tb(3+) is 9-coordinated with three water molecules and six OH groups from two D-galactitol molecules. Nitrate ions do not coordinate to metal ions, which is different from other reported lanthanide nitrate-D-galactitol complexes. In TbGN(II) and SmGN (Ln(NO(3))(3)·C(6)H(14)O(6)), Ln(3+) is 10-coordinated with six OH groups from two D-galactitol molecules and four oxygen from two bidentate nitrate ions, and one nitrate ion is hydrogen bonded. No water exists in the structures. D-Galactitol molecules provide their 1-, 2- and 3-hydroxyl groups to coordinate with one metal ion and their 4-, 5- and 6-hydroxyl groups to coordinate with another metal ion in the three structures. There is still a new topological structure that can be observed for lanthanide-d-galactitol complexes, which indicates that the coordinations between hydroxyl groups and metal ions are complicated.     

Chiral recognition of ethers by NMR spectroscopy

Enantiomers of chiral ethers and acetals are notoriously difficult to differentiate because their reactivity is low and they are poor donors to any Lewis acid or metal ion. As an exception, epoxides are somewhat better donors. This review describes the properties of ethers, explains NMR methods for their chiral recognition and describes successful examples of ether differentiation. The majority of literature reports deals with chiral lanthanide shift reagents and dirhodium tetracarboxylate complexes, which were used as enantiopure auxiliaries to create diastereomeric adducts with dispersed (1)H and (13)C NMR signals. The various methods are compared as to which is best suited for which purpose.     

Excited state properties of lanthanide complexes: Beyond ff states

Generally, metal-centered ff states dominate the discussion of the excited state properties of lanthanide complexes. In particular, the luminescence properties of Eu(III) and Tb(III) compounds have been studied in great detail for many decades. However, other types of excited states such as MC fd, MLCT, LMCT, MMCT and IL are also of interest. In this context, we have recently examined the excited state behavior of selected Ce(III), Ce(IV), Eu(II) and Gd(III) complexes which are luminescent and/or photoreactive.     

The light switch effect upon the association of [Ru(1,10-phenanthroline)2dipyrido[3,2-a:2',3'-c]phenazine]2+ with single stranded poly(dA) and poly(dT)

Large enhancement in the luminescence intensity of the Delta- and Lambda-Ru(phenanthroline)(2)dipyrido[3,2-a:2',3'-c]phenazine](2+) ([Ru(phen)(2)DPPZ](2+)) complexes upon their association with single stranded poly(dA) and poly(dT) is reported in this work. As the mixing ratio ([[Ru(phen)(2)DPPZ](2+)]/[DNA base]) increases, the luminescence intensity increase in a sigmoidal manner, indicating that the enhancement involves some cooperativity. At a high mixing ratio, the luminescence properties are affected by the nature of the DNA bases and not by the absolute configuration of the [Ru(phen)(2)DPPZ](2+) complex, indicating that the single stranded poly(dA) and poly(dT) do not recognize the configuration of the metal complex. In the case of the Lambda-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex, the manner of the enhancement is somewhat different from the other Ru(II) complex-polynucelotide combinations: the luminescence intensity reached a maximum at an intermediate mixing ratio of 0.32, and gradually decreased as the mixing ratio increased. In contrast to other complexes at high mixing ratios, an upward bending curve was found in the Stern-Volmer plot, which indicates that the micro-environment of the Lambda-[Ru(phen)(2)DPPZ](2+) is heterogeneous. In the Delta-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex case, formation of this highly luminescent species at an intermediate mixing ratio is far less effective.     

Antineoplastic activity of new lanthanide (cerium, lanthanum and neodymium) complex compounds

Cerium (III), lanthanum (III) and neodymium (III) complexes with 3,3'-benzylidenebis[4-hydroxycoumarin] were synthesized in view of their application as cytotoxic agents. The complexes were characterized by different physicochemical methods: elemental analysis, mass spectrometry, 1H NMR, 13C NMR and IR spectroscopy. The spectra of the complexes were interpreted on the basis of comparison with the spectrum of the free ligand. The vibrational analysis showed that in the complexes the ligand coordinated to the metal ion through both deprotonated hydroxyl groups; however, participation of the carbonyl groups in the coordination to the metal ion was also suggested. The evaluation of the cytotoxic activity of the novel lanthanide complexes on HL-60 myeloid cells revealed that they are potent cytotoxic agents. The cerium complex was found to exhibit superior activity in comparison to the lanthanum and neodymium coordination compounds, the latter being the least active. Our data give us reason to conclude that the newly synthesized lanthanide complexes should be submitted to further more detailed pharmacological and toxicological evaluation.     

Determination of the solution conformation of dephospho coenzyme A by nuclear-magnetic-resonance spectroscopy with lanthanide probes. A method for analysis when more than one complex species is present

The aqueous solution conformation of the 1 : 1 complex of dephospho CoA bound to a lanthanide ion has been determined by examination of the dipolar shift and induced relaxation at pH 6.4. The experimental data are shown to arise from the presence of both 1 : 1 and 1 : 2 metal : ligand complexes and a graphical method is described to divide the experimental data into information corresponding to each of the two species. The formation constants are also derived. For the 1 : 1 complex the ribose is found in a 2E conformation with the adenine base predominantly anti. A small contribution from a syn conformation is evident. The pantoinic acid fragment of the chain is folded back towards the pyrophosphate while the remainder of the chain is extended.     

Time-resolved immunofluorometric assays with measurement of a europium chelate in solution: application for sensitive determination of fibronectin

A novel detection principle applicable for sensitive measurement of molecules of biological interest by time-resolved fluorescence spectrophotometry is described. Our method is based on the quantification of the Eu3+ chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in solution in presence of an excess of Eu3+ ions. BCPDA-labeled solid phase complexes obtained by conventional immunoassay procedures are transferred into solution using urea/SDS/Eu3+ as dissociating and fluorescent lanthanide ion reagent. Two 'sandwich-type' assay variants based on the above methodology were realized for the determination of small amounts of fibronectin (FN) in biological fluids. FN is captured from solution by solid phase coated gelatin or a monoclonal antibody, respectively. Rabbit anti-FN antiserum used as second antibody is detected with a biotinylated anti-rabbit IgG antibody. Fluoresence is measured after incubation with streptavidin-BCPDA and dissociation of solid phase complexes as described. Both assays have a detection limit (blank + 3 x SD) of less than 0.5 ng/ml FN, a dynamic range of up to 300 ng/ml, and intraserial coefficients of variation of 4.4 and 6.3%, respectively. Median FN concentrations in saliva of healthy individuals were 104 (gelatin) and 36 ng/ml (double antibody), respectively.     

Application of "blue native" electrophoresis in the studies of mitochondrial respiratory chain complexes in physiology and pathology

"Blue Native" polyacrylamide gel electrophoresis (BN-PAGE), originally described by Sch?gger and von Jagow in 1991, is an elegant method to study protein complexes from mitochondrial membranes. BN-PAGE, commonly used in molecular biology to study composition of protein complexes and protein-protein interactions, enables separation of respiratory chain complexes keeping their properties and enzymatic activities unchanged. BN-PAGE, supplemented by other methods, e.g. in gel activity assay, SDS-PAGE (as a first or second dimension) can be successfully adapted for diagnosis of mitochondrial diseases connected with abnormalities of the respiratory chain. Therefore, to make a correct diagnosis of the deficiency of respiratory chain complexes, other methods, as histochemical colorimetric reactions allowing evaluation of the OXPHOS catalytic activity in individual cells and spectrophotometric technique should be used simultaneously with BN-PAGE.