Lanthanide Chelates Research Articles

Labeling of proteins and oligopeptides with luminescent lanthanide(III) chelates

Synthesis of a building block that allows introduction of photoluminescent europium(III) and samarium(III) chelates to synthetic oligopeptides on solid phase using standard Fmoc chemistry is described. Upon completion of the oligopeptide synthesis, these conjugates were converted to the corresponding lanthanide(III) chelates by treatment with appropriate lanthanide(III) salt. Also synthesis of a new terpyridine-based europium(III) chelate designed for solution phase protein labeling is demonstrated.

希土類錯体をドープしたＤＮＡ‐脂質複合体の光学特性

A DNA-lipid complex, where cationic surfactants are bonded to DNA, was synthesized and its optical property was examined by doping rare-earth chelates. The DNA lipid complex films to which several kinds of rare-earth chelates were doped were prepared, and their fluorescence quantum yields were examined. A 2 to 10-fold increase in quantum yield was observed as compared with conventional PMMA similarly doped. The improvement of the fluorescence quantum yield indicates that the use of DNA is promising in the development of new optical functional materials.

Targeting of lanthanide(III) chelates of DOTA-type glycoconjugates to the hepatic asyaloglycoprotein receptor: cell internalization and animal imaging studies

The characterization of a new class of hydrophilic liver-targeted agents for gamma-scintigraphy and MRI, consisting, respectively, of [(153)Sm](3+) or Gd(3+) complexes of DOTA monoamide or bisamide linked glycoconjugates (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), is reported. In vitro studies show high uptake of radiolabeled [(153)Sm]-DOTAGal(2) by the human hepatocyte carcinoma cell line Hep G2 containing the asialoglycoprotein receptor (ASGP-R), which is decreased to less than 50% by the presence of its high-affinity ligand asialofetuin (ASF). In vivo biodistribution, gamma-imaging and pharmacokinetic studies on Wistar rats using the [(153)Sm](3+)-labeled glycoconjugates show a high uptake in the receptor-rich organ liver of the radiolabeled compounds containing terminal galactosyl groups, but very little uptake for those compounds with terminal glycosyl groups. Blocking the receptor in vivo reduced liver uptake by 90%, strongly suggesting that the liver uptake of these compounds is mediated by their binding to the asyaloglycoprotein receptor (ASGP-R). This study also demonstrated that the valency increase improves the targeting capability of the glycoconjugates, which is also affected by their topology. However despite the specific liver uptake of the radiolabeled galactose-bearing multivalent compounds, the animal MRI assessment of the corresponding Gd(3+) chelates shows liver-to-kidney contrast effects which are not significantly better than those shown by GdDTPA. This probably results from the quick wash-out from the liver of these highly hydrophilic complexes, before they can be sufficiently concentrated within the hepatocytes via receptor-mediated endocytosis.

Formation constants of some ternary lanthanide(III) chelates with vitamin U and oxine

Formation of 1: 1: 1 ternary complex species (MAL) has been inferred and the relevant equilibrium has been established from pH measurements on the interaction of M 3+ ions [M = La III , Pr III , Sm III and Gd III ] with ligand A [HA = oxine] in the presence of the ligand L [HL = vitamin U] in aqueous medium at ionic strength of 0.2 and at varying temperatures 25 °C and 35 °C.

Extrinsic lyoluminescence of aluminum induced by lanthanide chelates in alkaline aqueous solution

Reactive Al/OH −(aq) interface was studied and used as a source of chemical energy in the generation of chemiluminescence. The observed extrinsic lyoluminescence emission during dissolution of aluminum in an alkaline Tb(III) or Eu(III) chelate solution was clearly based either on 5D 4→ 7F J radiative transitions of Tb(III) or 5D 0→ 7F J transitions of Eu(III). In this process, these chelates were chemically excited via analogous one-electron redox pathways as known from extrinsic lyoluminescence of irradiated, electrolytically- or additively-colored alkali halides, and from hot electron-induced electrochemiluminescence. Calibration curves of Tb(III) chelates, peroxodisulfate and hydroxide ions were linear over several orders of magnitude of concentration. In addition, the method seems to be suitable for relatively rough chemical measurements of the thicknesses of aluminum oxide films free from trapped charges.

A New Class of Macrocyclic Lanthanide Complexes for Cell Labeling and Magnetic Resonance Imaging Applications

Lanthanide complexes have wide applications in biochemical research and biomedical imaging. We have designed and synthesized a new class of macrocyclic lanthanide chelates, Ln/DTPA-PDA-C(n), for cell labeling and magnetic resonance imaging (MRI) applications. Two lipophilic Gd3+ complexes, Gd/DTPA-PDA-C(n) (n = 10, 12), labeled a number of cultured mammalian cells noninvasively at concentrations as low as a few micromolar. Cells took up these agents rapidly and showed robust intensity increases in T1-weighed MR images. Labeled cells showed normal morphology and doubling time as control cells. In addition to cultured cells, these agents also labeled primary cells in tissues such as dissected pancreatic islets. To study the mechanism of cellular uptake, we applied the technique of diffusion enhanced fluorescence resonance energy transfer (DEFRET) to determine the cellular localization of these lipophilic lanthanide complexes. After loading cells with a luminescent complex, Tb/DTPA-PDA-C10, we observed DEFRET between the Tb3+ complex and extracellular, but not intracellular, calcein. We concluded that these cyclic lanthanide complexes label cells by inserting two hydrophobic alkyl chains into cell membranes with the hydrophilic metal binding site facing the extracellular medium. As the first imaging application of these macrocyclic lanthanide chelates, we labeled insulin secreting beta-cells with Gd/DTPA-PDA-C12. Labeled cells were encapsulated in hollow fibers and were implanted in a nude mouse. MR imaging of implanted beta-cells showed that these cells could be followed in vivo for up to two weeks. The combined advantages of this new class of macrocyclic contrast agents ensure future imaging applications to track cell movement and localization in different biological systems.

Bimodal System (Luminophore and Paramagnetic Contrastophore) Derived from Ln(III) Complexes Based on a Bipyridine-Containing Macrocyclic Ligand

The synthesis of a new 15-membered polyaza-macrocyclic ligand L3H3, which is based on a 2,2'-bipyridine moiety and a diethylenetriaminetriacetic acid core, is reported. The lanthanide chelates of this octadentate ligand were programmed for bimodal probes, luminescent agents (Sm, Eu, Tb, Dy), and magnetic resonance imaging agents (Gd3+). The neutral 1:1 complexes with these Ln3+ ions were prepared and studied in aqueous solution by luminescence and NMR techniques. The main photophysical characteristics of these complexes (i.e., the absorption and luminescence spectra, the metal-centered lifetimes, and the overall luminescence yields, Phi) were measured. In addition, the role played by nonradiative pathways (vibrational energy transfer involving coordinated water molecules, involvement of ligand-to-metal charge-transfer excited states, or metal --> ligand back transfer) is discussed. The L3.Eu and L3.Tb complexes show very bright luminescence when photoexcited from the lowest-energy absorption band of the bipyridine chromophore. The luminescence quantum yields in an air-equilibrated water solution at room temperature are 0.10 and 0.21, respectively, despite the presence of one water molecule in the first coordination sphere of the metal ion. NMR data show that L3.Gd contains also one H2O molecule in the inner sphere. The proton longitudinal relaxivity, r1, of this complex is 3.4 s(-1) mM(-1) (0.47 T, 310 K) and the rotational correlation time, tau(R), is 57 ps (310 K). These values are comparable to those of the clinically used Gd-DTPA. Interestingly, the water exchange rate between the coordination site and the bulk solvent is slow (tau(M) = 3.5 micros at 310 K). The presence of water molecules in the second sphere and in rapid exchange with the solvent is discussed. Finally, it was found by luminescence and NMR experiments that these lanthanide complexes are stable versus transmetalation by several cations (especially Ca2+ and Zn2+) at physiological pH and have no interaction with blood proteins.

Homogeneous assay based on low quantum yield Sm(III)-donor and anti-Stokes’ shift time-resolved fluorescence resonance energy-transfer measurement

Non-overlapping fluorescence resonance energy-transfer (nFRET) is a highly sensitive, time-resolved, assay technology. It utilizes lanthanide chelates as donors together with non-overlapping acceptor fluorophores, which have their absorption maximum energetically at a higher level than the emittive transitions of the donor. In this report the nFRET was studied using a low quantum yield Sm(III)-chelate as the donor in a homogeneous dF508-DNA hybridization assay. Despite the low quantum yield, Sm(III) could function as an efficient donor in nFRET, which resulted in strong energy-transfer induced acceptor emission in the assay. Further, the induced acceptor emission of the assay showed non-Förster-type decay characteristics and allowed the sensitive anti-Stokes’ shift FRET measurement, in which the induced acceptor emission was measured at a wavelength region shorter than the main emittive transitions of the donor. The detection limit for the dF508 DNA-target was 12.0 pM (S/B = 2). The assay sensitivity was surprisingly similar with the corresponding Eu-based nFRET-assay, despite the approximately 90-fold lower quantum yield of the Sm-donor. The results confirm the exceptional properties of the nFRET induced acceptor signal and support the theory of energy-transfer taking place from the excited energy levels above the emittive energy level of the lanthanide donor. We conclude that nFRET-technique is well suited for highly sensitive DNA assays and that the assay sensitivity is not limited by the emittive quantum yield of the donor.

Analysis of Indian blue ballpoint pen inks tagged with rare-earth thenoyltrifluoroacetonates by inductively coupled plasma–mass spectrometry and instrumental neutron activation analysis

Characterization and assessment of inks on sensitive documents for absolute/relative age determination is the challenging forensic problem in spite of practical difficulties. Tagging of ballpoint pen ink with suitable taggant(s) is a unique method to come out with definitive inferences on the detection of forgery in documents written with ballpoint pens. Selection of a proper taggant primarily depends on sensitivity of analytical determination and their absence in normal varieties of ink used for document writing. Rare-earth elements, from all technical considerations can be potential taggant(s) for inks. To ensure more compatibility with ink, 13 rare-earth thenoyltrifluoroacetonate chelates were prepared and characterized. The ballpoint pen inks were tagged with rare-earth thenoyltrifluoroacetonate chelates individually at about 1–100 ppm level depending on sensitivity of element under suitable optimized experimental conditions and instrumental sensitivity. Aliquots of such tagged ink having varying amounts of taggants were analyzed by ICP–MS and INAA. Satisfactory recoveries and a good linear relationship of intensity (signal) against concentrations/amounts were observed. Under the optimized experimental conditions, the detection limits were worked out. This study of tagging metal ions in combination with ICP–MS and NAA as an analytical tool can allow to draw various combination options based on different rare-earth chelates as suitable materials for tagging of ballpoint pen inks for absolute/relative age determination to aid in document related crime examination. The advantages and limitations of proposed analytical techniques are discussed.

Progress in Lanthanides as Luminescent Probes

Lanthanides have recently found applications in different fields of biomolecular and medical research. Luminescent lanthanide chelates have created interest mainly due to their unique luminescent properties, such as their long Stokes' shift and exceptional decay times allowing efficient temporal discrimination of background interferences in the assays, such as immunoassays. Recently, new organometallic complexes have been developed giving opportunities to novel applications, in heterogeneous and homogeneous immunoassays, DNA hybridization assays, high-throughput screening as well as in imaging. In addition, encapsulating the chelates into suitable matrix in beads enables the use of new members of lanthanides extending the emission wavelength to micrometer range and decays from a few microseconds to milliseconds. As the luminescence is derived from complicated intra-chelate energy transfer, it also gives novel opportunities to exploit these levels in different types of energy transfer based applications. This review gives a short overview of recent development of lanthanide chelate-labels and discusses in more details of energy levels and their exploitation in new assay formats.

Emerging Applications of Phosphorescent Metalloporphyrins

The subject of phosphorescent metalloporphyrins is reviewed, focusing mainly on the development and application of Pt- and Pd-porphyrins. A summary of their general chemical and photophysical properties, and guidelines for rational design of the phosphorescent labels, bioconjugates and probes is given. Examples of different detection formats and particular bio-analytical applications developed in recent years are presented. The potential of phosphorescent porphyrin label methodology is discussed and compared to that of the long-decay fluorescent lanthanide chelates and other common fluorophores.

Lanthanide(III) Chelates of DTPA Bis(amide) Glycoconjugates: Potential Imaging Agents Targeted at the Asyaloglycoprotein Receptor

AbstractThe synthesis and characterisation of a new class of DTPA bis(amide) linked glycoconjugates of different sugars [lactose (Lac) and galactose (Gal)] and valencies (di and tetra) and their LnIII complexes is reported. The 1H NMR spectra of the SmIII and EuIII complexes of DTPAGal2, DTPAGal4, and DTPALac2, obtained between 7 and 80 °C, indicate that several of the four possible diastereoisomeric pairs of structures resulting from the chirality of the three bound DTPA nitrogen atoms are present in solution, with different relative populations. The dynamic effects of racemisation of the central nitrogen atom in the NMR spectra show that this process is fast at 60 °C for the SmIII complexes and slow at 7 °C for the EuIII complexes. The in vitro r1 nuclear magnetic relaxation dispersion (NMRD) of the water protons of the GdIII–DTPA bis(amide) glycoconjugate containing two lactosyl moieties [GdIII–DTPALac2] was studied, yielding the molecular parameters that govern its relaxivity. Its r1 value, at 25 °C and 20 MHz, is 13 % higher than that reported for GdIII chelates of lower molecular weight DTPA bis(amides), such as DTPA‐BMA, consistent with a five times larger τR value. The water exchange rate, kex, and the electron spin relaxation parameters of the GdIII–DTPALac2 complex are within the usual range for similar GdIII–DTPA bis(amide) chelates. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Solid-Phase Synthesis of Oligonucleotides Labeled with Luminescent Lanthanide(III) Chelates

The synthesis of phosphoramidite building blocks that allow introduction of luminescent europium(III), terbium(III), dysprosium(III), and samarium(III) chelates to oligonucleotides on the solid phase is described. Several labeled oligonucleotides using these building blocks were prepared, and the photophysical properties of these bioconjugates were investigated.

Simultaneous Use of Time-Resolved Fluorescence and Anti-Stokes Photoluminescence in a Bioaffinity Assay

A bioaffinity assay is described where anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide chelates are measured from a single microtitration well without any disturbance from these label technologies to each other. Up-converting phosphor (UPC-phosphor) bioconjugate was produced by grinding the commercial, micrometer-sized UPC-phosphors to colloidal, submicrometer-sized phosphor particles and by attaching these phosphors to biomolecules. Experiments were carried out in standard 96-well microtitration plates to determine detection limits, linearity, and cross-talk of UPC-phosphor and europium chelate. In numbers of molecules the lower limits of detection for UPC-phosphor were roughly 3 x 10(3) particles in solution and 1 x 10(4) particles in solid phase, and for europium label same values were 9 x 10(6) and 9 x 10(7) molecules. Linearity of detection was for UPC-phosphor 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase and for europium label over 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase. The cross-talk between the two labels was practically nonexistent. In this study we show that up-converting anti-Stokes photoluminescent phosphors could be employed in bioaffinity assays as very potential labels with significant advantages either alone or together with long-lifetime lanthanide chelates.

Synthesis, Characterization, and Application of Eu(III), Tb(III), Sm(III), and Dy(III) Lanthanide Chelate Nanoparticle Labels

Preparation and characterization of europium(III), terbium(III), samarium(III), and dysprosium(III) polystyrene nanoparticle labels with lanthanide-specific fluorescence properties has been presented. Emulsion copolymerization of styrene and acrylic acid was used to synthesize uniform-sized nanoparticles approximately 45 nm in diameter. Europium(III) and samarium(III) lanthanides were chelated with 2-naphthoyltrifluoroacetone and trioctylphosphine oxide to dye the spherical particles, whereas terbium(III) and dysprosium(III) chelate complexes contained a newly synthesized ligand, 4-(2,4,6-tridecyloxyphenyl)pyridine-2,6-dicarboxylic acid. The fluorescence properties of the four lanthanides-including a wide Stokes shift, a narrow emission peak, and long fluorescence lifetime-were retained despite the incorporation into the nanoparticles. Furthermore, the nanoparticles, containing more than 1000 lanthanide chelates, were detectable at label concentrations 3 orders of magnitude lower than the corresponding soluble lanthanide chelate labels. The applicability of the labels prepared was demonstrated by a heterogeneous sandwich-type immunoassay for human prostate-specific antigen, where the lowest limits of detection of 1.6, 2.4, 10.1, and 114.2 ng/L were achieved using europium(III), terbium(III), samarium(III), and dysprosium(III) nanoparticles, respectively. The spectral and functional properties of the lanthanide-embedded polystyrene nanoparticles developed here suggest that the technology is applicable for high-sensitivity multicolor assays.

Synthesis and Structural and Biological Activity Studies on Some Lanthanide Chelates with O‐ and N‐Containing Ligands

The synthesis of Ce3+, Th4+, and UO2 2+ complexes with six azo compounds based on 4‐methyl‐6,7‐dihydroxy‐8‐arylazo coumarin is reported. The azo compounds were expected to coordinate through O and N either in the azo or in the hydrazone form, but different physicochemical studies (elemental and thermal analysis, molar conductance, IR, magnetic susceptibility, 1H‐NMR, and electronic absorption spectra) show that they coordinate via O, O with the coumarins acting as mono (or sometimes di‐) basic and bidentate ligands. Biological activity of some complexes has been evaluated against some filamentous microscopic fungi, yeast, and bacteria. The results are compared to those of the free ligands.

Rare-Earth Quinolinates: Infrared-Emitting Molecular Materials with a Rich Structural Chemistry

Near-infrared-emitting rare-earth chelates based on 8-hydroxyquinoline have appeared frequently in recent literature, because they are promising candidates for active components in near-infrared-luminescent optical devices, such as optical amplifiers, organic light-emitting diodes, .... Unfortunately, the absence of a full structural investigation of these rare-earth quinolinates is hampering the further development of rare-earth quinolinate based materials, because the luminescence output cannot be related to the structural properties. After an elaborate structural elucidation of the rare-earth quinolinate chemistry we can conclude that basically three types of structures can be formed, depending on the reaction conditions: tris complexes, corresponding to a 1:3 metal-to-ligand ratio, tetrakis complexes, corresponding to a 1:4 metal-to-ligand ratio, and trimeric complexes, with a 3:8 metal-to-ligand ratio. The intensity of the emitted near-infrared luminescence of the erbium(III) complexes is highest for the tetrakis complexes of the dihalogenated 8-hydroxyquinolinates.

Ionic complexation properties of per(3,6-anhydro)cyclodextrin derivatives towards lanthanides

Using per(3,6-anhydro)cyclodextrin derivatives [per(3,6-anhydro)CD], it was possible to produce new lanthanide chelates by careful choice of the size and functional groups. Heptakis(3,6-anhydro-2- O-methyl)cyclomaltoheptaose fulfils the best criteria for complexation of lanthanide ions. Nuclear magnetic resonance was used to derive the association constants and the stoichiometries of these new complexes. Finally, a three-dimensional structure of these complexes consistent with the NMR data is proposed, to ascertain the position of lanthanide in the cavity of the per(3,6-anhydro)CD. For the present purposes, heptakis(2- O-acetyl-3,6-anhydro)cyclomaltoheptaose, octakis(2- O-acetyl-3,6-anhydro)cyclomaltooctaose, heptakis(3,6-anhydro-2- O-methyl)cyclomaltoheptaose and octakis(3,6-anhydro-2- O-methyl)cyclomaltooctaose have been synthesized and purified.

Detection scheme for bioassays based on 2,6-pyridinedicarboxylic acid derivatives and enzyme-amplified lanthanide luminescence

2,6-Pyridinedicarboxylic acid (PDC) and its derivatives are introduced as a new sensitizer system for enzyme-amplified lanthanide luminescence (EALL), a detection scheme for bioassays, which combines enzymatic amplification with time-resolved luminescence measurements of lanthanide chelates. Various PDC esters have been synthesized as esterase substrates that are cleaved to PDC in the presence of the enzyme. PDC forms luminescent complexes with Tb(III) or Eu(III), and the evaluation of the reaction is used for the selective and sensitive detection of esterases. For an esterase from hog liver a limit of detection of 10 −3 u/mL (equivalent to 10 −9 mol/L) and a limit of quantification of 3 × 10 −3 u/mL (equivalent to 3 × 10 −9 mol/L) could be achieved. As a second model reaction, xanthine oxidase (XOD) catalyzes the oxidation of 2,6-pyridinedicarboxaldehyde to PDC. Here, the limit of detection was 3 × 10 −3 u/mL and the limit of quantification 10 −2 u/mL for XOD from microorganisms. Major advantage of the tridentate PDC ligand is the possibility to perform all steps of the assay within or close to the physiological pH range, while the established EALL schemes based on bidentate salicylates or bisphenols have to be carried out at strongly alkaline pH to ensure sufficient complexation with the lanthanides.

Homogeneous time-resolved fluorescence quenching assay (TruPoint) for nucleic acid detection.

Laboratories performing genetic screening studies need simplified methods that allow automation for nucleic acid analysis. Ideally, a method should have as few handling steps as possible and allow the use of a closed-tube assay format to reduce the risk of PCR contamination. Different homogeneous assay chemistries have been exploited to integrate amplification and detection to allow simple nucleic acid analysis with minimal post-PCR handling (1)(2)(3)(4)(5). Double-stranded DNA probes consisting of a 5′-terminus-labeled fluorescent strand and a complementary strand labeled on the 3′ terminus with a quencher have been exploited in homogeneous hybridization (2)(6)(7). Fluorescence signal is generated when the fluorophore strand hybridizes with the target. When the quencher strand hybridizes with the fluorophore strand, the signal from the fluorophore is quenched. Morrison et al. (6) used probes consisting of strands of equal length and a competitive hybridization technology, whereas others have exploited probes consisting of strands with different lengths for detection of PCR products (2)(7). The use of stable and fluorescent lanthanide chelates has enabled the development of homogeneous assay technologies based on time resolution (8). Time-resolved fluorescence-quenching assays (TR-FQAs), based on energy transfer from a lanthanide chelate to a nonfluorescent quencher, have been applied in various assays of hydrolyzing enzymes (9)(10) and are commercialized as TruPoint™. The first homogeneous PCR assays with lanthanide-labeled probes used an environmentally sensitive terbium chelate, the fluorescence intensity of which changes on probe hybridization (3)(11). Subsequently, the technology was further improved by use of additional quencher probes (12). Recently, a combination of quenching and competitive hybridization has been applied for end-point detection of PCR products (13). The aim of the present study was to compare two homogeneous PCR assay approaches based on a …