Luminescent Labels Research Articles

Time-resolved, total internal reflection fluorescence microscopy of cultured cells using a Tb chelate label

A total internal reflection fluorescence microscope with time-resolved spectroscopy and CCD imaging capabilities is described. The capability of the microscope to selectively detect, via temporal resolution, the presence of a long-lived luminescence label in biological cells in vitro was evaluated. Tb chelates having radiative lifetimes of ca. 1.5 ms were employed as long-lived, extrinsic labels. Microspectroscopy and imaging were performed on mouse 3T3 cells stained with Tb chelates, stained with DiI (an extrinsic membrane label of ns lifetime), and in the absence of an extrinsic label. The results demonstrate the efficiency of using time-resolved detection to discriminate against intrinsic and extrinsic sources of short-lived emission in cultured cells, which enables long-lived, extrinsic luminescence to be selectively detected. Potential applications of this technology are discussed.

Luminescent Visualization of Low Amounts of Cytochrome P450 and Hemoproteins by Luminol in Acrylamide Gels

In this paper we apply luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), a light-emitting substrate, in conjunction with H2O2 to the luminescence labeling of hemoproteins. We describe in detail a photodetection device which permits an efficient recording of the light emitted by heme-containing proteins resolved in acrylamide gels. The sensitivity of this procedure, when compared to the classical 3,3′-5,5′-tetramethylbenzidine staining method, results in a 5- to 20-fold enhancement with standard hemoproteins (lactoperoxidase, catalase, cytochrome P450 2B4, horseradish peroxidase, hemoglobin, myoglobin, and cytochrome b5). The potential applications of this technique are illustrated by the detection of cytochrome P450 in microsomes from plant as well as from animal extrahepatic tissues which possess low amounts of this cytochrome.

Luminescent Labels for Purine Nucleobases: Electronic Properties of Guanine Bound to Rhenium(I)

Luminescent Labels for Purine Nucleobases: Electronic Properties of Guanine Bound to Rhenium(I)

ChemInform Abstract: Luminescent Labels, More than Just an Alternative to Radioisotopes?

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Luminescent Labels—More than Just an Alternative to Radioisotopes?

AbstractChemical, chromatographic, or spectrometric methods are generally unsuitable for the detection of molecules in the nano and subnanogram region because of their low sensitivity. The radioimmunoassay (RIA) developed by Yalow and Berson in 1959 combined the high sensitivity of radioactively labeled substances with the high specificity of immunological reactions for the first time. In this way it was possible to detect quantitatively the tiniest traces of substances in the presence of an excess of other, in some cases, similar foreign substances without prior enrichment. Immunoassays have certainly developed to become the most valuable analytical tool of in vitro diagnostics and are today routinely employed for the detection of endogenous and exogenous substances (e.g. hormones, tumor‐associated proteins, bacteria, viruses, toxins, drugs, etc). The many disadvantages of radioactivity such as the required handling licenses, disposal costs, precautions necessary to prevent risks to health, short shelf‐life, and limited sensitivity soon led to the search for other nonradioactive labeling methods. Encouraged by the development of light measuring techniques and the commercial availability of highly sensitive apparatus, radioactive isotopes as labels are today being replaced increasingly by enzymes, fluorophores, or luminophores. Some of the new luminescent labels have, however, not only facilitated replacement of radioisotopes, but also a breakthrough into what has until now been unattainable levels of sensitivity. The following article reviews the methods of luminescent labeling and their applications mainly in the area of immunoassays.

UV-visile absorption and fluorescence characteristics of the luminescent label coumarin-6-sulphonyl chloride in homogeneous and micellar solutions

This paper describes the study of the UV-visual absorption, Fluorescence emission, and fluorescence excitation spectra of coumarin-6-sulphonyl chloride (C-6SCl) in various organic solvents and buffer solutions. In addition, the spectral behaviour in aqueous micellar systems of anionic and cationic surfactant was investigated. The spectral properties of the compound together with the detemined p K values in the ground and excited state are discussed in relation to its electronic structure. The results reflect the importance of the medium effect on the analytical application of C-6SCl as a potential luminescent label for derivatization of amino and phenolic groups.

Complexes of lanthanoid salts with macrocyclic ligands. 41. Photophysical properties of lanthanide dinuclear complexes with p-tert-butylcalix[8]arene

The macrocyclic octaphenol p-tert-butylcalix[8]arene (LH[sub 8]) was reacted with lanthanide(III) ions in dimethylformamide (DMF) containing triethylamine to obtain both homo- and heterodinuclear neutral complexes of composition [(Ln1:Ln2)LH[sub 2](DMF)[sub 5]](DMF)[sub n], with n = 4 ([alpha]-phase) or 1.5 ([beta]-phase). ICP-AES determination of the Ln(III) content shows a clear selectivity of the ligand for ions in the middle of the lanthanide series. Luminescence measurements at 77 K suggest that the two lanthanide(III) ions encompassed by the ligand are in very similar environments with pseudo C[sub 3h] symmetry. Small differences in the crystal-field potential are evidenced between the two crystalline phases and when a large ion (e.g Nd) is encapsulated by the ligand. The presence of a low-lying metal-to-ligand charge-transfer state (MLCT) in the Eu-containing complexes at ca. 20,000 cm[sup [minus]1] induces unusual spectroscopic properties. Very large absorption probabilities ([approximately]10[sup [minus]6]) have been determined for the Eu(III) transitions, and the Judd-Ofelt theory for f-f transitions fails to explain the very large value of the [Omega][sub 2] parameter (448 x 10[sup [minus]20] cm[sup 2]). In DMF solution, an efficient energy transfer from the ligand to Tb(III) occurs and makes the Tb(III) calixarene complex an interesting luminescent label.

Luminescence studies of polyelectrolyte behaviour in solution—I. Accessibility of naphthalene-based labels of poly(methacrylic acid) to mobile low molar mass species in aqueous media

A novel approach to the study of the conformational behaviour of water-soluble polymers is reported. The method is based upon the generation of stabilized, room temperature phosphorescence using mobile “heavy-atom” promoters (Tl +). The conjunct roles of phosphorescence intensity and lifetime data in providing information upon polyelectrolyte behaviour is illustrated, using samples of poly(methacrylic acid) labelled with 1-vinylnaphthalene or acenaphthylene as luminescent labels. The resultant data are compared with those obtained using more conventional fluorescence methodologies.

Encapsulated lanthanide ions as efficient luminescent species

Results dealing with the photophysical properties of complexes of the Eu 3+ and Tb 3+ ions with encapsulating ligands are reviewed. It is shown that such compounds are species capable of emitting a strong, long-lived luminescence in water solution. The use of these compounds as luminescent labels is described. Selected examples are discussed in an attempt to analyze the factors that determine the luminescence intensity.

116 Establishment and evaluation of immunometric assays for the determination of human ferritin in serum using enzyme, luminescent and time-resolved fluorescent labels

116 Establishment and evaluation of immunometric assays for the determination of human ferritin in serum using enzyme, luminescent and time-resolved fluorescent labels

Inorganic phosphors as new luminescent labels for immunocytochemistry and time‐resolved microscopy

A new strongly luminescent marker consisting of inorganic crystals is described for time-resolved microscopy. These crystals, known as phosphors, show delayed luminescence, unlike prompt fluorescent labels such as FITC, TRITC and phycobiliproteins, and are therefore potentially suitable for time-resolved microscopy. The luminescence of these phosphors is strong and non-fading in comparison to FITC/TRITC, and not significantly influenced by pH or temperature. The phosphor yttriumoxisulfide activated with europium emits maximally at 620 nm with a typical half life-time of approximately 700 musec, upon excitation with near ultraviolet light (360 nm). Phosphors for immunocytochemical staining were made by ball milling and were stabilized in suspension with polycarboxylic acids. Proteins such as avidin, protein A or immunoglobulins were allowed to adsorb to the surface of the phosphors. The immunocytochemical properties of the conjugates were evaluated in a model system of latex beads with defined surface antigens and in a cellular system containing fixed human lymphocytes or erythrocytes. Specific cytochemical staining was observed in suspension as well as on glass slides. A specially constructed time-resolved microscope was used to suppress the fast decaying fluorescence, thereby permitting visualization of the specific, slowly decaying luminescence of the phosphor label without the necessity of integration. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.

Luminescent labels for immunoassay—from concept to practice

Luminescent labels for immunoassay—from concept to practice

Chemiluminescence immunoassay for the detection and quantification of methylestosterone residues in muscle tissue

Chemiluminescence as a detection method for immunoassays has succesfully been applied to the measurement of methyltestosterone (MT) residues in muscle tissue. The sample is digested enzymatically, extracted with diethyl ether and purified on a Lipidex-5000 column. An optional clean-up utilized disposable C 18 columns. As the luminescent label the N-(4-aminobutyl)-N-ethylisoluminol conjugate of MT was used. The antiserum was raised in a rabbit against MT-3-carboxymethyloximebovine serum albumin. The detection limit of the assay was 14 ± 7 pg ( n = 13), with a limit of quantification in muscle tissue of 0.125 ppb.

Interaction of Cu(II) ions with poly(4-vinylpyridine) studied by luminescence quenching

Interaction of Cu(II) ions with poly(4-vinylpyridine) carying luminescent labels was studied in aqueous methanol by means of the luminescence quenching method. The dependence of fluorescence intensity on the concentration of the quencher (cupric ions) does not obey the Stern-Volmer equation. An analysis of this experimental dependence led to the conclusion that the distribution of metal ions between the macromolecules is not uniform. The kinetics of the exchange of metal ions between occupied and free macromolecules was investigated.

Interphase chromatin of peripheral blood cells in patients with chronic myeloid leukemia (Ph+)

These distinguishing features of the curves mentioned above (evidently indicating a change in the structural organization of chromatin) in patients with inborn chromosomal anomalies [3, 5] and correlations of these deviations with these characteristics of the curves in the separate subgroups of normal individuals (as possible genetic predisposition) make it essential to analyze the structural features of cell chromatin in other chromosomal aberrations also. In the investigation described below an analysis of this kind was undertaken on peripheral blood cells of patients with an acquired chromosomal anomaly, namely chronic myeloid leukemia, CML (Ph+), during periods of blast crisis and clinical and hematologic compensation. EXPERIMENTAL METHOD Nuclear chromatin of peripheral blood cells of 25 patients with CML (Ph § aged between 16 and 60 years in different stages of the disease was studied. Changes in chromatin structure during heating (from 20 to 95~ were recorded as the quantity of luminescent label acridine orange (AO) bound after every I-2.5~ The investigations were conducted on cells incubated for 1 h in Eagle's nurtrient medium with the addition of 10% auto!ogous serum.

Luminescence immunoassays applied to routine measurements of plasma proteins

The aim of this presentation is to demonstrate that luminescence immunoassays for serum proteins can be introduced as routine laboratory methods. The small sample volume maximally 20 ~tl for ferritin makes the assays ideal for paediatric use. The serum proteins chosen were: ferritin, transferrin and caeruloplasmin as metalloproteins, orosomucoid, c-reactive protein and transthyretin as acute-phase proteins and ~l-foetoprotein as "tumour marker" and pregnancy risk parameter. The methods used were variations of solid-phase chemiluminescent immunoassays developed in this laboratory, namely SPALT (solid-phase antigen luminescence techniques) [1] and ILSA (immunoluminometric labelled second antibody assay), both of which used labelled second (species-specific) antibody, and ILMA (immunoluminometric assay) which used a labelled substance-specific (first) antibody, analogous to the immunoradiometric assay (IRMA). All assays had one reaction partner coupled covalently to a polystyrene ball (6.4ram diameter) as solid phase. The luminescent label adapted for all assays was N-(4-aminobutyl)-N-ethyl isoluminol hemisuccinamide (ABEI-H) which was commercially available (LKB, Mfinchen). This was converted into an N-hydroxysuccinimide ester, which was then coupled to the antibody in an analogous way to the BoltonHunter iodination of proteins. All assays were measured in either an LKB-1251 25 sample luminometer (LKB, Mfinchen) or an LB-950 300 sample luminometer (Labor Prof. Dr. Berthold, Wildbad), the latter being more suitable for assay runs with more than about 30 samples. Table1 shows the assay conditions, reference ranges in serum and relevant quality-control parameters for each assay. This article has demonstrated that luminescence immunoassays are fully capable of being used routinely, and need no longer be seen as "exotica" of little or no practical value!

ChemInform Abstract: SYNTHESIS OF FLUORESCENT PROBE ‐ CARBOHYDRATE CONJUGATES

AbstractCarbohydrate Conjugates.

Synthesis of fluorescent probe – carbohydrate conjugates

A new type of luminescent label has been synthesized and a range of carbohydrates has been derivatized with various fluorescent probes. Amine-containing labels were readily transformed with chloroacetylchloride into the corresponding fluorescent chloroacetamide reagents 1–4. Suitably blocked monosaccharides containing either a free thio 9, hydroxyl 11, or carboxyl 17 functionality have been conjugated with (a) 4-chloroacetamido labels 1 and 2, (b) with 4-aminobenzophenone 5, and (c) with 1-dimethyl-1-aminonaphthalene-5-sulfonylchloride 21, to form the fluorescent carbohydrate conjugates 10, 16, 18, and 20. Direct derivatization of the unblocked disaccharides 22 and 23 was achieved by reductive amination with amines 5 and 7 and sodium cyanoborohydride affording the 1-deoxyglycit-1-yl derivatives 24–26, which were characterized by 13C nmr spectroscopy. The synthesis of fluorescent polysaccharides is exemplified by the condensation of 9-anthraldehyde with chitosan either via Schiff 's base formation to afford 32, or via reductive alkylation to form 33.

Synthesis routes for phenyl containing polymers with anthracene groups

Polarized luminescence was used to study the relaxation characteristics of phenyl containing polymers (PCP) which were produced with luminescence labels on the anthracene structure; these are in special parts of the macromolecules (the branch groups, the main chain, or at the chain ends). The methods for producing PCP with anthracene groups are based on free radical polymerization in which anthracene containing monomers or chain terminating agents participate.