Harri Takalo

Correlation between the lowest triplet state energy level of the ligand and lanthanide(III) luminescence quantum yield

Abstract The luminescence properties of 41 different Eu(III) and Tb(III) chelates that were synthesized with the purpose of developing new markers for chemical and biochemical applications were measured in aqueous solution and their suitability for labels in time-resolved immunoassays were evaluated. In spite of the influence of numerous factors on the luminescence quantum yields, a clear correlation was obtained between the energies of the lowest triplet state levels of the ligands and lanthanide (III) luminescence quantum yields of the respective chelates. Simultaneously Eu(III) and Tb(III) ions were observed to accept energy by their several different 5Dj, resonance levels depending on the energy of the lowest triplet state level of the ligand, and the Tb(III) luminescence quantum yields were observed to decrease due to the energy back transfer from excited Tb(III)∗ to the ligand when the energy difference between the 5D4 level of Tb(III) and the lowest triplet state energy level of the ligand is less than 1850 cm−1.

Immunoassay by time-resolved electrogenerated luminescence

Abstract An immunoanalytical method based on electrogenerated luminescence was developed in which the labelling compound is the terbium chelate of 2,6-bis[ N,N -bis(carboxymethyl)aminomethyl]-4-benzoylphenol which is linked by a thioureido group to the antibody. The sandwich complex of the labelled antibody, antigen and antibody immobilized on the surface of an aluminium electrode is excited by an electrical pulse and, after a short delay from the end of the pulse, light emission is measured. The electroluminescence immunoassay is exemplified by the heterogeneous and homogeneous assays of human pancreatic phospholipase A 2 , for which both assay methods give a reasonably good linearity in the range 10–300 ng ml −1 .

Development of luminescent lanthanide chelate labels for diagnostic assays

Abstract Luminescence properties of some of the lanthanide ions form the basis of highly sensitive diagnostic methods which employ their chelates as probes and time resolved fluorometry in the detection. In addition to being highly luminescent, i.e., having an efficient light collection and energy transfer, photostability, good quantum yield and long excited state lifetime, an ideal luminescent chelate label must be small and highly hydrophilic and contain a functional group enabling its gentle coupling to the bioreagent. Certain qualifications have to be fulfilled when luminescent chelate labels are to be made, such as the optimal position of ligand triplet states, position and type of the binding arm, insulation of the coupling group from the energy absorbing moiety, a critical distance between the luminescent moiety and protein surface (in case of Tb) and elimination of all possible quenching routes associated with O-H and C-H stretching (in case of Eu). Lanthanide chelates have already proven to be very useful in highly sensitive immunoassays and simplified homogeneous assays. In addition, the new chelates developed permit materialization of numerous future ideas, such as miniaturized assay devices able to perform multi-parametric assays on sub-microliter volumes of samples.

Synthesis and luminescence properties of some Eu(III) and Tb(III) chelate labels having 2,2′:6′,2″-terpyridine as an energy absorbing part

Abstract A series of luminescent 2,2′:6′,2″-terpyridine lanthanide chelates, in which the group needed for coupling to bioactive molecules is situated in the chelating part of the molecule, was synthesized. The luminescence properties of the labels both as free activated chelates and after coupling to proteins were also studied.

Tb(III) chelate chemiluminescence induced by the dissolution of additively coloured potassium chloride in aqueous solutions of peroxydisulphate ions

Abstract Dissolution of an additively coloured alkali halide crystal in an aqueous peroxydisulphate solution results in the formation of hydrated electrons and sulphate radicals. These radicals are capable of inducing terbium(III) chelate chemiluminescence by fast subsequent redox reactions, where the radiative 5D4 → 7Fj transitions of coordinated terbium(III) is based on a chemical excitation of the aromatic ligand by its interactions with these radicals, which is followed by an intramolecular transfer of this excitation energy to the 5D4 state of terbium(III).

A time-resolved study of the mechanism of the energy transfer from a ligand to the lanthanide(III) ion in solutions and solid films.

The photochemical properties of some lanthanide chelates developed for immunohistochemistry have been studied in water and in solid Langmuir-Blodgett films. The fluorescence and triplet-state lifetimes of 4-(phenylethynyl)pyridine (PET), di[(phenylethynyl)pyridine] (D-PET), phenylterpyridine (PTP) and their tetra- or penta-acid derivatives (-TA or -PA) were measured in the presence and absence of Gd(III)-, Tb(III)- and Eu(III)-ions. The mechanism for the total process and the rate constants and quantum yields for the individual reaction steps and for the total process were determined in water solution. Time-resolved absorption and luminescence methods were also used to study the energy transfer between an amphiphilic 4-[4-[(C(10)H(12))(2)NCO]phenylethynyl]-pyridine tetra acid (A-PET-TA) and the Tb(III)- and Eu(III)-ions in solid Langmuir-Blodgett films. Luminescence and transient absorption rate constants were determined.

Evaluation of solution structures of highly luminescent europium(III) chelates by using laser induced excitation of the 7F0 → 5D0 transition

Solution structures of 13 Eu(III) chelates were examined by using laser induced excitation of the 7F0 → 5D0 transition. Remarkable variations in the 7F0 → 5D0 excitation spectra of 2,2′,2″,2″−{[aryl]bis(methylenenitrilo}tetrakis(acetic acid) complexes of Eu(III) are observed depending on the denticity of the ligand and the number and character of the coordinated nitrogen atoms. The evaluation of the structures is made on the basis of the energy of the 7F0 → 5Do excitation transition of Eu(III) because the 7F0 → 5D0 transition energy is dependent on the number and type of coordinating atoms in the first coordination sphere of Eu(III). Additional information about the structures is obtained by measuring the excited-state lifetimes of the Eu(III) chelates. The 7F0 → 5D0 transition energy shifts always an equal amount to lower energies due to the coordination of a certain group or atom. The energies of the 7F0 → 5D0 excitation transitions are also used to calculate these ‘nephelauxetic’ shift parameters for coordinated nitrogen heteroatoms iin the 2,2′,2″,2″−{[4-phenylethynyl)pyridine-2,6-diyl]bis (methylenenurido)}tetrakis(acetic acid) (3). 2,2′,2″,2″-[(2,2′-bipyridine-6,6′-diyl)bis-(methylenenitrilo)] tetrakis (acetic acid) (4). 2,2′,2″,2″-[(2,2′:6,2″-terpyridine-6,6″-terpyridine-6.6″-diyl)bis(methylenenitrilo)]tetrakis(acetic acid) (5), 2,2′,2″,2″- (pyrazole-1,3-diyl)-bis(pyridine)-2,2′-diyl]bis(methylenenitrilo)]tetrakis(acetic acid) (7). 2,2′,2″,2″-([thizole-2,4-diyl)bis(pyridine)- 2,2′-diyl]-bis(methylenenitrilo(]tetrakis(acetic acid) (8) and 2,2′,2″,2″-([2,2,′-pyridine-2,6-diyl)bis(thiazole)-4,4′-diyl]bis-(methylenenitrilo)]-tetrakis(acetic acid) (9) complexes. The variation in the shift parameters of the nitrogen heteroatoms is suggested to be due to the different distances between the nitrogen heteroatoms and Eu(III) ions.

Enhanced EuIII ion luminescence and efficient energy transfer between lanthanide chelates within the polymeric structure in aqueous solutions

Four new binucleating symmetric bis(4-pyridine-2,6-dicarboxylic acids) have been synthesized and luminescence enhancement of their EuIII complexes by YIII ions observed. The results indicate that the mechanism of enhancement is based on the formation of a polymeric structure where EuIII ions and YIII ions are linked together by the ligands with the consequent transfer of absorbed energy along the polymer chain through several ligands toward the EuIII ion. The mechanism of energy transfer is probably different from the previously reported micellar co-luminescence where energy transfer occurs via the Forster dipole–dipole mechanism.

Improved surface stability and biotin binding properties of streptavidin coating on polystyrene.

The ultimate nature of streptavidin to bind biotin tightly is widely utilized in many solid-phase based applications to provide a universal binding surface for biotinylated molecules. However, the preparation of the streptavidin coatings by passive adsorption may heavily alter the binding properties of native streptavidin and may not result in the best possible capture surface for demanding solid-phase assays. By introducing sulphydryl groups through primary amines in the protein, we have activated and conjugated native streptavidin into larger protein polymers resulting in high local binding density when coated on polystyrene. This thiolated streptavidin formed through chemical modification has improved adsorption properties and biotin binding capability, compared to the native streptavidin. When this thiolated streptavidin is coated on polystyrene, a dense surface is formed, which provides up to 3-fold increase in the biotin binding efficiency and improves the surface stability by minimizing the desorption of the adsorbed protein from the surface during incubation. Furthermore, this high-capacity surface is resistant to harsh chemical treatments, such as denaturing conditions or mild reducing conditions. The improved adsorption properties of the thiolated streptavidin allow the coating process to be performed with shorter incubation times (15min), still providing enhanced solid-phase properties, compared to a reference streptavidin surface.

Homogeneous duplex polymerase chain reaction assay using switchable lanthanide fluorescence probes.

We have developed a duplex polymerase chain reaction (PCR) assay based on switchable lanthanide chelate complementation probes. In the complementation probe technology, two nonfluorescent oligonucleotide probes, one labeled with a lanthanide ion carrier chelate and another with a light absorbing antenna ligand, form a fluorescent complex by self-assembly of the reporter molecules when the two probes are hybridized in adjacent positions to the target DNA. Here we report the synthesis of a new terbium(III) (Tb(III)) ion carrier chelate and a new light-absorbing antenna ligand for Tb(III) and the development of a duplex Chlamydia trachomatis (Ct) PCR assay. For the detection of Ct in urine samples, a specific sequence in Ct cryptic plasmid was amplified and detected using europium(III) (Eu(III)) complementation probes. An internal amplification control was amplified in each reaction and detected using Tb(III) complementation probes to verify the Ct negative results. Ct bacteria were concentrated from urine samples with a rapid and simple centrifugation-based sample preparation method. Good diagnostic accuracy (99-100%) was achieved, and also Ct positive reactions yielded a very high Eu(III) signal-to-background ratio (maximum of 244). High performance of the complementation probes is advantageous when sample may contain impurities after a simple sample preparation.