Terbium Chelates Research Articles

Tetravalent Terbium Chelates: Stability Enhancement and Property Tuning.

Coordination chemistry of rare-earth elements has been dominated by the +3 oxidation state. Complexes with higher-valence lanthanide ions are synthetically challenging but are of fundamental research interest and significance as advanced molecular materials. Herein, four tetravalent terbium complexes (2-5) of the common formula [Tb(OSiPh3)4L] (L = ethylene glycol dimethyl ether (DME), 2,2'-bipyridine (bpy), 2,2'-bipyrimidine (bpym), and 1,10-phenanthroline (phen)) are reported. Crystallographic analyses reveal in each of these complexes a hexacoordinate Tb(IV) ion situated in a distorted octahedral coordination environment formed by four triphenylsiloxido ligands and a bidentate chelating ligand. The use of chelating ligands enhances the stability of the resulting complexes over their THF solvate precursor. More significantly, the aromatic N-chelating ligands have been found to tune effectively the electronic structures of the complexes, as evidenced by the sizable potential shifts observed for the quasi-reversible redox Tb(IV/III) process and by the changes in their absorption spectra. The experimental findings are augmented with quantum theoretical calculations in which the ligand π-donation to the 5d orbitals of the Tb(IV) center is found to be primarily responsible for stability enhancement and the corresponding changes of physical properties observed. Magnetic measurements and results from electron paramagnetic resonance studies produced small absolute values of zero-field splittings of these complexes, ranging from 0.1071(22) to 1.1484(112) cm-1 and comparable to the values reported for analogous Tb(IV) complexes.

Terbium(III) as a Fluorescent Probe for Molecular Detection of Ascorbic Acid

Fluorescence analysis is a simple and a highly sensitive method for detection of small amounts of biologically active substances. In this study, a complexation of terbium(III) chelates with 1,10-phenanthroline and ascorbic acid (AA) and luminescent properties of complexes were investigated. The influence of pH and solubilization of complexes by micellar solutions of nonionic, cationic, and anionic surfactants on fluorescence was studied. The quenching effect of terbium ion fluorescence was detected upon an introduction of ascorbic acid. The quenching effect of the complex with mixed ligands Tb(1,10-phenanthroline)-AA allows for the detection of ascorbic acid with the limit of 7.4 × 10−5 mol·L−1.

Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity.

This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

A Limited 4 Å Radial Displacement of the S4-S5 Linker Is Sufficient for Internal Gate Closing in Kv Channels

Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.

Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Förster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1Å. Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was logY=3.65786+0.43863·logX (R=0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0.4ngmL−1. By assaying test samples against the standard curve, the coefficient of variations was <5%, indicating that QDs were suitable for this homogenous time-resolved fluoroimmunoassay. This work extended the potential applications of QDs in future homogeneous analytical bioassays. In the coming research, hepatitis B surface antigen, another primary marker for hepatocellular carcinoma, will be studied for practical detection using a QD-based homogenous multiplex fluoroimmunoassay.

Effects of terbium chelate structure on dipicolinate ligation and the detection of Bacillus spores

Terbium-sensitized luminescence and its applicability towards the detection of Bacillus spores such as anthrax are of significant interest to research in biodefense and medical diagnostics. Accordingly, we have measured the effects of terbium chelation upon the parameters associated with dipicolinate ligation and spore detection. Namely, the dissociation constants, intrinsic brightness, luminescent lifetimes, and biological stabilities for several Tb(chelate)(dipicolinate) x complexes were determined using linear, cyclic, and aromatic chelators of differing structure and coordination number. This included the chelator array of NTA, BisTris, EGTA, EDTA, BAPTA, DO2A, DTPA, DO3A, and DOTA (respectively, 2,2′,2″-nitrilotriacetic acid; 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N,N′,N′-tetraacetic acid; 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid; diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid; 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Our study has revealed that the thermodynamic and temporal emission stabilities of the Tb(chelate)(dipicolinate) x complexes are directly related to chelate rigidity and a ligand stoichiometry of x = 1, and that chelators possessing either aromaticity or low coordination numbers are destabilizing to the complexes when in extracts of an extremotolerant Bacillus spore. Together, our results demonstrate that both Tb(EDTA) and Tb(DO2A) are chemically and biochemically stable and thus applicable as respectively low and high-cost luminescent reporters for spore detection, and thereby of significance to institutions with developing biodefense programs.

Multiparametric luminescence method for quantitative cell surface protein expression analysis and imaging

A luminometric method for quantitative cell surface protein expression analysis has been developed in a microtiter plate format. The method is based on immunocytochemistry, the use of long-lived europium(III) and terbium(III) chelates and platinum(II) porphyrin luminescence labels in addition to short-lived syto13 DNA stain, and detection of photoluminescence emission from adhered cells by both time-resolved luminescence and conventional fluorescence. After the immunoreactions, the wells were evaporated to dryness, allowing repeated and postponed luminescence analysis even after months and cellular protein localization studies by microscopy imaging. The multiparametric method assayed the cell surface expression of ß1-integrin, E-selectin and intercellular adhesion molecule 1 (ICAM-1) in HUVE cells (human umbilical vein endothelial cells). The expression of E-selectin and ICAM-1 was enhanced by treating HUVECs with tumor necrosis factor α (TNF-α), while the expression level of ß1-integrin remained unchanged. The sensitivity limit of TNF-α detection by the method was ca. 1pg/ml and the Z′-factors for the quantification of E-selectin and ICAM-1 were >0.7 suggesting a highly robust method. The novel approach proposed in this paper can be potentially applied to cell surface protein expression analysis in screening applications combined with localization studies of the target proteins by fluorescence microscopy imaging.

Comparison of Homogeneous Single-Label Fluorometric Binding Assays: Fluorescence Polarization and Dual-Parametric Quenching Resonance Energy Transfer Technique

The time-resolved fluorescence technique, quenching resonance energy transfer, QRET, relies on a single-labeled binding partner in combination with a soluble quencher. The quencher reduces efficiently the fluorescence of the unbound labeled ligand, whereas the fluorescence of the bound fraction is detectable. This approach allows the development of homogeneous screening assays in a simple and cost-effective manner. In this study, two single-label fluorometric methods, fluorescence polarization (FP) and the QRET technique, are compared in a simple biochemical model immunoassay of estradiol. Estradiol-6-amino was labeled with fluorescein and lanthanide(III) chelates for the FP and QRET assays, respectively. The labeled estradiols were allowed to compete against free estradiol, and the assay parameters were investigated. The EC(50) value of QRET assay using europium(III)-labeled estradiol was 0.1 nM, and the assay sensitivity was approximately 10 pM. These values were more than 10-fold lower than those for the FP assay with Z' values higher than 0.75 for both assays. The high sensitivity was attributed to a low concentration of antibody fragment and labeled estradiol used in the QRET assay. This reduces cost in screening studies without sacrificing the assay performance. A signal-to-background ratio (S/B) of more than 20 was reached in the QRET assay at elevated concentration of the assay components, whereas S/B of 3.6 was measured in the FP assay when both assays shared the same EC(50) value of 1 nM. Multiplexing of assays is a cost-effective means to run screening studies as multiple data can be extracted from a single experiment. Therefore, multiplexing of the QRET assay was investigated and its feasibility was successfully demonstrated in a dual-parametric assay using estradiol labeled with europium(III) and terbium(III) chelates.

Luminescent Terbium Protein Labels for Time‐Resolved Microscopy and Screening

Brilliance of terbium: Heterodimeric conjugates of trimethoprim covalently linked to sensitized terbium chelates bind to Escherichia coli dihydrofolate reductase fusion proteins with nanomolar affinity (see picture). Terbium luminescence enables sensitive and time-resolved detection of labeled proteins in vitro and on the surface of living mammalian cells.

Detection of Quadruplex DNA by Luminescence Enhancement of Lanthanide Ions and Energy Transfer from Lanthanide Chelates

Small amounts of quadruplex DNA have been detected using luminescence enhancement of aqueous lanthanide ions and energy transfer from lanthanide chelates. The 22mer human telomeric DNA, AGGG(TTAGGG)(3), was detected using europium ions at concentrations as low as 20 ppb DNA. Detection with terbium ions was not possible due to the inherent weak luminescence intensities of lanthanides. Two different terbium chelates were used to overcome this challenge. When quadruplex DNA was added to the chelates there was a change in the excited-state lifetime of the chelate with subsequent energy transfer to the DNA. Experiments showed an increase in the amount of energy transferred from the chelate to the human telomeric DNA and other quadruplex sequences increased as a function of DNA concentration.

Facile Conversion of FP to TR-FRET Assays using Terbium Chelates:Nuclear Receptor Competitive Binding Assays as Examples

Time-resolved FRET (TR-FRET) assays offer advantages over fluorescence polarization (FP) assays including reduced optical interference from compound autofluorescence or precipitation, and a greater ability to resolve potencies of tight-binding ligands. Here, we demonstrate the facile conversion of FP assays to a TR-FRET format using nuclear receptor ligand binding assays as examples.

Sensitive Terbium Probes for Luminescent Determination of both Alkaline Phosphatase and Codeine Phosphate

New terbium complexes of 2-oxo-4-hydroxy-quinoline-3-carboxylic acid derivatives are reported for determination of alkaline phosphatase (aPase) and codeine phosphate (CP). Complexation with terbium occurs by way of the oxygen atoms of the 4-hydroxyl group and the carbonyl group of the 3-carboxy function, respectively. The complexes are highly luminescent and do not require luminescence enhancers. The excitation maxima of the terbium chelates are at 320 nm. The assay is based on the quenching of the hypersensitive 545-nm luminescence of the terbium complexes with the ligands 4-hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid (5-ethyl-[1,3,4]thiadiazol-2-yl)-amide (L(1)) and 1-ethyl-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid (4-trifluoromethyl-phenyl)-amide (L(2)) by phosphate ions. The assays require equimolar (1.0 microM) concentrations of Tb(3+) and of the ligand at pH 8.0 (Tris-HCl buffer). The luminescence quenching is proportional to the concentration of aPase and CP within the range of 0.1-70 mU mL(-1) and 0.3-20 microg mL(-1), respectively. The detection limits were 0.05 mU mL(-1)=40.0 pmol L(-1) of aPase and 0.120 microg mL(-1) of CP.

An automated PCR platform with homogeneous time-resolved fluorescence detection and dry chemistry assay kits

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.

Luminescent energy transfer between cadmium telluride nanoparticle and lanthanide(III) chelate in competitive bioaffinity assays of biotin and estradiol

Fluorescence resonance energy transfer has been studied between lanthanide(III) chelates as donors and protein-coupled CdTe semiconductor nanoparticles as acceptors. Wide excitation spectra and large Stokes shift of semiconductor nanoparticles and timeresolved fluorescence detection were shown to provide a combination for successful energy transfer assay. Different intrinsically fluorescent europium(III) and terbium(III) chelates coupled to single biotin molecules were studied for optimal energy transfer with streptavidin labeled semiconductor nanoparticles. No significant differences between the studied chelates were observed. The strength of the methodology was demonstrated in a clinically relevant competitive and separation-free immunoassay of estradiol, where subnanomolar limit of detection was achieved with the coefficient of variation 2–11%. The data suggested that relatively short distance was needed to obtain adequate energy transfer. Therefore, biomolecules were coupled onto the semiconductor nanoparticles without any spacers.

Simultaneous Monitoring of Discrete Binding Events Using Dual-Acceptor Terbium-Based LRET

Lanthanide resonance energy transfer (LRET) between chelated terbium and two spectrally distinct acceptor fluorophores (fluorescein and Alexa Fluor 633) is demonstrated. Ligand-dependent association and dissociation of differentially labeled peptides derived from coactivator and corepressor proteins with PPAR isoforms was monitored simultaneously, with no mathematical deconvolution required to separate the signals from the distinct acceptor fluorophores. These results demonstrate the utility of terbium chelates for the analysis of complex multicomponent binding events by LRET.

Convenient Synthesis of Maleimido-Derivatized Lanthanide(III) Chelates and Their Use in Mercapto Group Conjugation

Simple synthesis of luminescent europium(III) and terbium(III) chelates tethered to a maleimido function (7, 8) is described. The method is based on the following: (i) synthesis of protected ligands tethered to a maleimido function and their purification on silica gel; (ii) deprotection by acidolysis; (iii) conversion of the deprotected ligands to the corresponding lanthanide(III) chelates by passing them through a column of strong cation exchange resin loaded with the appropriate lanthanide(III) ions. According to this procedure, large quantities of mercapto-selective biomolecule-labeling reactants of high purity can be prepared.

Determination of some hydroxybenzoic acids and catechins in white wine samples by liquid chromatography with luminescence detection

A liquid chromatographic method with luminescence detection for the determination of eight phenolic compounds is reported. The method involves postcolumn derivatization with terbium(III). This derivatization is based on the reaction between phenolics and terbium(III) to form luminescent chelates, which were determined at lamda ex 295 and lamda em 545 nm using the fluorescence mode. The long wavelength emission of lanthanide chelates can minimize interferences from background sample matrix, which usually emit at shorter wavelengths. Also, the chromatographic separation allows the individual determination of phenolics, which cannot be done using the direct measurement of the fluorescence of their corresponding terbium chelates. Dynamic ranges of the calibration graphs and detection limits, obtained with standard solutions of analytes were (microg/mL): gallic acid (0.9-40, 0.3), protocatechuic acid (0.05-7, 0.016), catechin (0.2-40, 0.07), vanillic acid (0.25-40, 0.08), p-hydroxybenzoic acid (0.8-40, 0.25), syringic acid (0.17-40, 0.05), epicatechin (0.3-40, 0.09) and salicylic acid (0.07-12, 0.02). The precision was established at two concentration levels of each analyte and expressed as the percentage of RSD with values ranging between 1.0 and 6.5%. The practical usefulness of the method was demonstrated by the analysis of white wine samples, which were diluted two-fold and directly injected into the chromatographic system. The recovery values obtained ranged between 93.3 and 108.0%.

Synthesis of Fluorescent Analogs of α-Conotoxin MII

Alpha-conotoxins (alpha-CTxs) are small peptides that are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) and have been used to study the kinetics of nAChRs. Alpha-CTx MII, from the venom of Conus magus, has been shown to potently block both rat alpha3beta2 and rat chimeric alpha6/alpha3beta2beta3 cloned nAChRs expressed in Xenopus oocytes. Tetramethylrhodamine (TMR), Bodipy FL, Alexa Fluor 488, and terbium chelates (TbCh) are fluorescent molecules that can be reacted with the N-terminus of the conopeptide to produce fluorescent conjugates. TMR and Bodipy FL were individually conjugated to alpha-CTx MII using different succinimidyl ester amine labeling reactions resulting in the formation of carboxamide conjugates. Alexa Fluor 488 succinimidyl ester conjugation reaction yielded low amounts of conjugate. TbCh was also individually reacted with the N-terminus of MII using the isothiocyanate conjugation reaction resulting in the formation of a thiourea conjugate. The conjugates were purified using reverse-phase high-pressure liquid chromatography (RP-HPLC) and their masses verified by matrix-assisted laser desorption-ionization with time-of-flight mass spectroscopy (MALDI-TOF MS). When tested on target nAChRs expressed in Xenopus oocytes, TMR-MII, Bodipy FL-MII, and TbCh-MII potently blocked the response to acetylcholine with slow off-rate kinetics. These fluorescent conjugates can be used to localize specific subtypes of neuronal nAChRs or ligand-binding sites within receptors in various tissue preparations; additionally, they may also be used to study conformational changes in receptors using fluorescence or lanthanide-based resonance energy transfer.

Temperature-dependent luminescent properties of Eu–Tb complexes synthesizedin situin gel glass

The chelates of europium and terbium with hexafluoroacetylacetone (HFA) and triphenylphoshine oxide (TPPO), Eu∕Tb(HFA)3(TPPO)2, have been synthesized in situ in gel glasses with various concentrations of Eu3+ and Tb3+ ions. The photoluminescence spectra have been measured and the characteristic transitions of Tb3+ and Eu3+ have been observed. Due to the variance of energy transfer efficiencies from Tb3+ to Eu3+, the intensity ratios of europium luminescent band to terbium band vary remarkably with measurement temperatures. In addition, the Förster mechanism has been proved to be responsible for the energy transfer between Eu3+ and Tb3+. The materials doped with Eu∕Tb(HFA)3(TPPO)2 are promising for being used as a temperature detector and thermal-sensitive probe of optical fiber sensor.

New luminescent terbium complex for the determination of DNA

New terbium complexes of derivatives of 2-oxo-4-hydroxy-quinoline-3-carboxylic acid are reported, which are highly luminescent, water soluble and do not require luminescence enhancers. The triplet-state energy levels of the ligands, the relative quantum yields (QYs) and the excitation maxima of the respective terbium chelates were determined. The large luminescence enhancement of one of these complexes by nucleic acids was investigated and a mechanism of its interaction with DNA is proposed. The optimal conditions for determination of DNA are equal concentrations of Tb 3+ and ligand R 1 ( C=1×10 −6 M), pH 9.0. Under optimal conditions the luminescence intensity (RI) is proportional to the concentration of fish sperm DNA (fsDNA) or calf thymus DNA (ctDNA), respectively, within the range of 0.05–1.5 μg ml −1. The detection limits were 10 ng ml −1 for fsDNA and 12 ng ml −1 for ctDNA.