Terbium Chelates Research Articles

Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer.

A powerful combination of molecular beacon and luminescence resonance energy transfer technology reveals alterations in nucleic acid structure by as little as a single nucleotide in a novel hybridization proximity assay. The assay measures the length of a single-stranded target when a terbium chelate-labeled molecular beacon hybridizes to one side of the nucleic acid segment to be measured and an acceptor probe carrying a convention fluorophore hybridizes to the opposite end of the target. Using a test sequence shortened incrementally by deleting single nucleotides, this assay reports a nearly linear relationship between sequence length and the distance separating acceptor and donor probes. Consequently, this assay can be used to detect alternative splicing, allele types, rearrangements, insertion, and deletion events by measuring separation distances within a predefined region. Furthermore, the use of terbium chelates in molecular beacons can produce exceptionally high signal-to-background ratios compared to the use of conventional fluorophores. Principles of optimal probe design are investigated experimentally and by computational simulations of plausible molecular beacon folding. Some molecular beacon designs form dimers that reduce their maximal response to target sequences. A simple assay to detect such dimers is reported as a tool to help improve the design of molecular beacons. Optimally designed molecular beacons with terbium chelates and hybridization proximity assays are expected to expand their applications in the analysis and screening of genetic diseases.

Element-coded affinity tags for peptides and proteins.

Isotope-coded affinity tags (ICAT) represent an important new tool for the analysis of complex mixtures of proteins in living systems [Aebersold, R., and Mann, M. (2003) Nature, 422, 198-207]. We envisage an alternative protein-labeling technique based on tagging with different element-coded metal chelates, which affords affinity chromatography, quantification, and identification of a tagged peptide from a complex mixture. As proof of concept, a synthetic peptide was modified at a cysteine side chain with either a carboxymethyl group or acetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (AcBD) chelates of terbium or yttrium. A mixture of the three modified peptides in a mole ratio of 100:1.0:0.83 carboxymethyl:AcBD-Tb:AcBD-Y was trypsinized, purified on a new affinity column that binds rare-earth DOTA chelates, and analyzed by LC-MS/MS. Chelate-tagged tryptic peptides eluted cleanly from the affinity column; the tagged peptides chromatographically coeluted during LC-MS analysis, were present in the expected ratio as indicated by MS ion intensity, and were sequence-identified by tandem mass spectrometry. DOTA-rare earth chelates have exceptional properties for use as affinity tags. They are highly polar and water-soluble. Many of the rare earth elements are naturally monoisotopic, providing a variety of simple choices for preparing mass tags. Further, the rare earths are heavy elements, whose mass defects give the masses of tagged peptides exact values not normally shared by molecules that contain only light elements.

Comparative study on fluorescence enhancement and quenching of europium and terbium chelate anions in cationic micelles

Fluorescence enhancement and quenching of water soluble chelates of terbium (Tb 3+) with Tiron, salicylic acid (SA), 4-sulfonyl salicylic acid (SSA) and acetylacetone (AA) and sparingly soluble chelates of europium (Eu 3+) with β-diketones were comparatively examined in the presence of cetyltrimethyl ammonium bromide (CTMAB) and cetylpyridinium chloride (CPC). By the composition of the complexes, surface tension measurements and spectral analysis, the binding mode of chelate anions to the micellar surface of cationic surfactants was discussed in terms of ion-exchange model. Quenching effect of CPC on the fluorescence of association complexes seems to arise from the charge transfer from a fluorescent ligand to pyridinium cation. In the case of the chelates of Eu 3+ with β-diketones, however, pyridinium ion is only capable of overlapping the aromatic ring of β-diketones to less extent since the poorly soluble charged chelates have a weak affinity for the highly polar surface of pyridinium cationic micelles. Efficient charge transfer between the excited aromatic β-diketone and pyridinium cation fails to be established. CPC also shows enhanced effect on fluorescence like CTMAB.

Characterization and structures of the 2,2,7-trimethyl-3,5-octanedionate chelates of cerium(IV) and terbium(III)

The complexes Ce(tod) 4 and Tb 2(tod) 6 were synthesized using hydrated metal nitrates and the β-diketone ligand H(tod), (tod=2,2,7-trimethyl-3,5-octanedione) with base neutralization. The metal complexes have been characterized by X-ray crystallography, elemental analysis, infrared spectroscopy, mass spectrometry, differential scanning calorimetry, and thermogravimetric analysis. The structure of Ce(tod) 4 represents a new polymorph for tetrakis cerium chelates and Tb 2(tod) 6 is the first reported dimeric β-diketonate structure for terbium. These materials are promising precursor sources of cerium and terbium for thin film deposition, and are highly soluble in supercritical carbon dioxide, making them surrogates for hazardous waste remediation using supercritical fluid extraction.

A homogeneous DNA hybridization system by using a new luminescence terbium chelate.

Homogeneous DNA hybridization assay based on the luminescence resonance energy transfer (LRET) from a new luminescence terbium chelate, N,N,N(1),N(1)-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA)-Tb(3+) (lambda(ex) = 325 nm and lambda(em) = 545 nm) to an organic dye, Cy3 (lambda(ex) = 548 nm and lambda(em) = 565 nm), has been developed. In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; one of the probes is labeled with the Tb(3+) chelate at the 3'-end, and the other is with Cy3 at the 5'-end. Labeling of the Tb(3+) chelate is accomplished via the linkage of a biotin-labeled DNA probe with the Tb(3+) chelate-labeled streptavidin. Strong sensitized emission of Cy3 was observed upon excitation of the Tb(3+) chelate at 325 nm, when the two probe DNAs were hybridized with the target DNA. The sensitivity of the assay was very high compared with those of the previous homogeneous-format assays using the conventional organic dyes; the detection limit of the present assay is about 30 pM of the target DNA strand.

Terbium-sensitized luminescence: a selective and versatile analytical approach

The special luminescent features of some terbium(III) chelates and the appropriate selection of experimental factors give rise to a wide variety of analytical applications such as direct determination of organic analytes, immunodiagnostic assays and chromatographic and electrophoretic methods, which are jointly presented and discussed in this review. The requirements for sensitizing terbium luminescence provide a high selectivity to these methods. Terbium-sensitized luminescence can be easily adapted to different analytical fields and shows a great analytical versatility.

Europium(III) and terbium(III) chelates of quinolonecarboxylic acid derivatives as labels for immunofluorimetric assay

Luminescence properties of Tb(III) and Eu(III) complexes of quinolonecarboxylic acid derivatives were studied. Optimal conditions of luminescence were determined, and the influence of surfactants and diethylenetriaminepentaacetic acid on the luminescence properties of the complexes was studied. It was demonstrated that species-specific immunoglobulins labeled with terbium ions can be determined with the detection limit of the lanthanide label 5 x 10-14 M.

Multiplex immunoassays on size-categorized individual beads using time-resolved fluorescence

Miniaturized multiplex immunoassays were studied on individual beads to detect analytes in the same reaction mixture. With this approach hands-on time, cost, and amount of reagents as well as waste produced in the assays were reduced. Particles were categorized according to size for immunoassays of prostate specific antigen (PSA) and acute myocardial infarction (AMI) markers. Additionally, free and dual PSA assays were carried out on a single bead. The analyte concentration was detected directly on the surface of the beads using stable, intrinsically fluorescent europium and terbium chelates, and time-resolved fluorometry. Less than 0.4ngml−1 PSA (corresponding to ca. 10amol) was detected in free, dual and multiplex PSA assays and 0.1 and 2.4ngml−1 of myoglobin (Mb) and creatine kinase MB (CK-MB), respectively, were monitored in the multiplex AMI marker assay. The effect of bead size and material on free PSA detection was also investigated. The beads were detected three times under different conditions; in liquid, after drying and after dissociating europium ions from the chelate into the DELFIA® fluorescence enhancement solution. The same detection sensitivity was found for the dissociative and non-dissociative methods indicating that the current labeling technology for the surface detection is comparable to the commercial DELFIA system.

Time-Resolved Detection and the Mechanism of Excitation of Soft X-Ray Irradiation-Induced Luminescence of Aliphatic Terbium(Iii) Chelates in Aqueous Solutions

Time-resolved detection utilizing a rotating chopper for x-rays allowed us die comparison between soft x-ray irradiation-induced luminescences (RL) and photoluminescences (PL) of TbIII aq complexed by chloride ion, sulfate ion, and different combinations of five aminopolycarboxylates. RL lifetimes and relative intensities of TbIII aq were observed to increase with the number of coordinating groups of the aliphatic ligand analogously to the PL results. No deviation from the Horrocks-Sudnick behaviour was observed. Experimental results support the proposal that the aliphatic complexes of TbIII aq are excited via direct action of radiation under soft x-ray irradiation.

Co-Fluorescence Enhancement System Based on Phenyl Salicylate and Lanthanum for the Determination of Terbium

A sensitive co-fluorescence enhancement system was developed for the fluorimetric determination of terbium. The fluorescence intensity of the chelates of Tb3+ with phenyl salicylate in a solution containing Triton X-100 and ethanol were greatly enhanced by the addition of excesses of La3+, Lu3+, Y3+ and Gd3+. The enhanced fluorescence intensity of the terbium chelates was 33, 20, 11 and 13 fold, respectively. At pH=9.0 the fluorescence intensity of the Tb3+ system with phenyl salicylate and La3+ is a linear function of concentration of Tb3+ in the range of 5.0 × 10−8 - 3.0 × 10−6 mol/L. The detection limit (signal-to-noise ratio = 3) is 3.0 × 10−9 mol/L.

Diagnostic imaging using rare-earth chelates

A new class of polyazamacrocyclic chelates of terbium is studied that have rich spectroscopic properties, tissue selectivity [1], millisecond fluorescence lifetimes, sharply spiked emission spectra ( 280 nm), good water solubility [1] and high fluorescence quantum yields (∼0.6 for PCTMB) [1]. We report our in vitro and ex vivo spectroscopic evaluation of these chelates. Additionally, results from cellular binding specificity studies using Sprague-Dawley rats with UMR 108 osteosarcomas are presented. Finally, endoscopic imaging of dosed tissue demonstrates the potential to use the Tb(III) chelates as contrast enhancement markers.

In situ molecular association of dystrophin with actin revealed by sensitized emission immuno-resonance energy transfer.

A novel method was developed to detect molecular associations of dystrophin with actin in cryostat muscle tissue sections by combining resonance energy transfer technology with immunohistochemical techniques. This method takes advantage of the long phosphorescent lifetime of terbium chelates, a property that enables the accurate determination of energy transfer in biological tissues by lifetime measurements of sensitized emission. After a brief excitation pulse, terbium chelates emit for milliseconds after the intrinsically high autofluorescence of biological specimens has decayed to negligible levels. Rat skeletal muscle tissue sections were labeled with both anti-dystrophin monoclonal antibody conjugated to a terbium-based resonance energy transfer donor and anti-actin tetramethylrhodamine phalloidin as an acceptor. Resonance energy transfer between the two probes indicated that the distance separating the probes is within 10 nm (about the size of an IgG2b antibody molecule). The fraction of antibodies that participated in resonance energy transfer was estimated to be 80-90% because of the close agreement between the quenching of donor phosphorescence and the efficiency of resonance energy transfer revealed by lifetime measurements of sensitized emission by tetramethyl-rhodamine phalloidin. Sensitized emission was detectable only when both anti-dystrophin antibody and tetramethyl-rhodamine phalloidin were present. These results indicate that actin and dystrophin are closely associated within the cell. This method is potentially applicable to the investigation of many types of intracellular associations.

Development of Luminescent Terbium(III) Chelates for Protein Labelling: Effect of triplet‐state energy level

AbstractThe synthesis of novel TbIII labels suitable for protein labelling are reported. Their luminescence properties as antibody conjugates were measured and compared to the results of corresponding TbIII chelates of the parent ligand structures. When the lowest triplet‐state energy level of the parent donor ligand was over 23000 cm−1, i.e., the energy gap between the 5D4 level of TbIII and the lowest triplet‐state energy level of the ligand exceeded 2600 cm−1, the label derivative with a long decay time (τ = 1.35–2.93 ms) and a high luminescence yield (ϵ. Φ = 3770–4560) was found to be suitable for bioaffinity assays.

Spectroscopic Characterization and Tissue Imaging Using Site-Selective Polyazacyclic Terbium(III) Chelates

Polyazamacrocyclic chelates of terbium are shown to be useful in diagnostic medical imaging as tissue site-selective markers. Spectroscopic properties and biodistribution are studied for three terbium(III) species: 3,6,9-tris(methylene phosphonic acid n-butyl ester)-3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca-1(15),11,13-triene (abbreviated as PCTMB); 3,6,9-tris(methylene phosphonic acid)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(13),11,13-triene (abbreviated as PCTMP); and N,N'-bis(methylene phosphonic acid)-2,11-diaza [3.3]-(2,6)pyridinophane (abbreviated as BP2P). The respective aqueous molar absorptivities are found to be 3424, 2513, and 3281/2210 L mole−1 cm−1. Fluorescence quantum efficiency is determined against rhodamine 19 in basic ethanol and rhodamine 6G in ethanol. These values are 0.48, 0.21, and 0.40 for Tb-PCTMB, Tb-PCTMP, and Tb-BP2P, respectively. Biodistribution studies performed in Sprague–Dawley rats indicate tissue site-selectivity. Fluorescence images of bone tissues are presented and demonstrate the potential for using the lanthanide chelates to perform site-directed in vivo imaging for the early identification of abnormal tissue.

Simultaneous high-performance liquid chromatographic determination of ochratoxin A and citrinin in cheese by time-resolved luminescence using terbium

A simultaneous reversed-phase HPLC determination of two major mycotoxins, ochratoxin A and citrinin, in soft cheese is proposed. Both mycotoxins are eluted on a C 18 RP support (25 × 4.6 mm I.D.) using an isocratic eluent consisting of methanol-water (70:30, v/v) containing tetrabutylammonium hydroxide (10 −3 M), acidified to pH 5.5 with HCl, and pumped at a flow-rate of 0.8 ml/min. Prior to detection, a butanolic solution of 5·10 −3 M terbium-5 · 10 −4 M trioctylphosphine oxide (TOPO)-2.5 · 10 −2 M triethylamine (TEA) was pumped in a postcolumn mode at a flow-rate of 0.2 ml/min to perform time-resolved luminescence (TRL) detection of the corresponding terbium chelates ( λ ex = 331 nm/ λ em = 545 nm). The method is linear from 3.5·10 −6 to 2·10 −5 M for citrinin and from 1·10 −5 to 5·10 −5 M for ochratoxin A. The repeatability and reproducibility (R.S.D.) are 1.9 and 2.4% for citrinin ( c = 3.5·10 −6 M; n = 10), and 7.2 and 8.3% for ochratoxin A ( c = 1.0·10 −5 M; n = 10). The limits of detection, for a signal-to-background ratio of 3, are 2·10 −6 and 3·10 −6 M for citrinin and ochratoxin A, respectively. With the proposed method, ochratoxin A and citrin are easily determined in soft cheeses, with a significative increase in selectivity in comparison with direct fluorescence detection.

Synthesis of rare‐earth metal‐containing polymers and their fluorescence properties

AbstractFour kinds of polymeric β‐diketone compounds, including poly(vinylbenzyldibenzoylmethane)(1), poly(vinylbenzylacetoacetone)(2), poly(vinylbenzyl‐2‐thenoyltrifluoroacetone)(3) and poly(p‐benzoylacetylstyrene)(4) were synthesized. And then polymeric β‐diketone‐europium(III), ‐terbium(III), ‐samarium(III), ‐dysprosium(III), ‐europium(III)‐gadolinium(III), ‐terbium(III)‐gadolinium(III), and ‐europium(III)‐terbium(III) chelates were also synthesized. The chelate structures were confirmed. The influence of the structure on the fluorescence properties was investigated. The fluorescence intensity of polymeric rare‐earth metal complexes is closely related to the ligand and metal content. Polymeric complexes possess a higher fluorescence stability than that of corresponding low‐molecular‐weight complexes.

Novel Thiazole‐Containing Complexing Agents and Luminescence of Their Europum(III) and Terbium(III) Chelates

AbstractThe synthesis of novel thiazole‐containing complexing agents and their luminescence properties with EuIII and TbIII ions are reported. One of these terpyridine analogues was also tested as an EuIII labelling reagent, and its luminescence properties as an antibody conjugate were studied.

X-ray irradiation-induced optical luminescence of terbium(III) chelates in aqueous solutions

Hydrated Tb(III) can be excited in aqueous solutions by various pathways during X-ray irradiation. The dominating pathway is probably due to chemical excitation, induced by oxidation of Tb(III) by ionized water molecules, followed by immediate reduction of the resulting Tb(IV) to Tb(III) by hydrated electrons or atomic hydrogen. The high enthalpy of this reduction reaction produces Tb(III) in its highly excited states resulting in typical 5 D 4 → 7 F i multiplet peak emissions. Other possible excitation pathways are direct excitation of Tb(III) by an impact of X-ray photons or photoelectrons, photoluminescent UV excitation due to the solid state emission of the cell materials, and/or an energy transfer from the excited water molecules. The same excitation mechanisms are also valid for chelated Tb(III), but the chelates with aromatic moieties produce luminescence predominantly by a sensitized mechanism where the aromatic moiety is first chemically excited by radical reactions, followed by an energy transfer to the central ion. Radiative relaxation of chelated Tb(III) results in an emission spectrum similar to that of hydrated Tb(III), but with a radioluminescence intensity considerably stronger than that of hydrated Tb(III).

Novel labelling agents for immunoassay by time-resolved electrogenerated chemiluminescence

In order to develop more efficient labelling agents for the binding assays based on time-resolved electrogenerated chemiluminescence, thirteen derivatives of (1-hydroxybenzene-2,6-diyl)bis)(methylenenitrilo)tetrakis(acetic acid) were synthesized. The aim was to study the influence of different substituents on the intensity of electrogenerated chemiluminescence (ECL) of their terbium(III) chelates. For comparison, also photoluminescent intensities of these chelates were recorded at the terbium emission line corresponding to the transition 5 D 4 → 7 F 5 (546 nm). The highest ECL intensities were recorded for the parent compound and its 4-methyl derivative, whereas the photoluminescent intensity was highest for the 4-cyano and 4-phenyl derivatives. There was not statistically significant correlation of the ECL intensity with the Hammett substituent constants.

Development of Luminescent Europium(III) and Terbium(III) chelates of 2,2′:6′,2″‐ terpyridine derivatives for protein labelling

AbstractThe synthesis and luminescence properties are reported for 20 different chelates composed of 2,2′:6′,2″‐terpyridine as the energy‐absorbing and donating group, EuIIIand TbIII as the emitting ions, methylenenitrilo(acetic acids) as the stabel chelate‐forming moieties, and isothiocyanato or(4,6‐dichloro‐1,3,5‐triazin‐2‐yl)amino groups as the activated moieties for coupling to biomolecules.