Journal of bioluminescence and chemiluminescence最新文献

The bioluminescent reaction catalysed by firefly luciferase has become widely established as an outstanding analytical system for assay of ATP. When used in solution, luciferase is unstable and cannot be re-used, a problem that can be partially circumvented by immobilizing the enzyme on solid substrates. Transparent glass is especially advantageous over alternative immobilizing matrices, since it allows most of the emitted photons to be detected. We report a new method for luciferase immobilization on glass which does not require prior silanization and glutaraldehyde activation, thus saving preparation time and minimizing enzyme inactivation. Our method is based on the co-immobilization by adsorption of luciferase (from a firefly lantern extract) and poly-L-lysine (PL) on non-porous glass strips. Luciferase immobilized in this way exhibits minimal variations in intersample activity, high sensitivity for ATP detection (linear luminescence responses down to 50 nmol/L) and good stability (full activity for at least 60 days when stored at -80 degrees C). PL-mediated immobilization of luciferase on glass strips provides an attractive strategy for the design of specific ATP biosensors, with potential in industry, environmental screening, medicine and biological research.

A cDNA encoding the Renilla reniformis luciferase was expressed in similan and murine cells in a transient and stable manner, respectively. Light emission catalyzed by luciferase was detected from transfected cells both in vitro and in vivo. This work establishes the Renilla luciferase gene as a new efficient marker of gene expression in mammalian cells.

4-Substituted phenyl boronic acids (e.g., 4-iodo, 4-bromo, 4-phenyl) are effective enhancers of the horseradish peroxidase (Type VIA) catalysed chemiluminescent oxidation of various pyrido[3,4-d]pyridazine-1,4(2H,3H)dione derivatives. The most effective combination was 4-biphenylboronic acid and 8-amino-5-chloro-7-phenylpyrido[3,4-d]- pyridazine-1,4(2H,3H)dione. Generally, the intensity of light emission in the presence of peroxidase was higher with the pyridopyridazines than with sodium luminol. However, the blank light emission was much lower with sodium luminol than with the pyridopyridazines. A synergistic enhancement phenomenon was demonstrated for the combination of a 4-iodophenol and a 4-biphenylboronic acid enhancer with 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H) dione. The combination of these two enhancers produced a light emission intensity in an assay for 5 fmol of peroxidase that was 25% higher than expected from the sum of the individual light intensities.

The reactions between superoxide free radical anion (.O2-) with the halocarbons CCl4. CHCl3, BrCH2CH2Br(EDB), decachlora-biphenyl (DCBP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dimethyl sulphoxide (DMSO) results in the emission of chemiluminescence (CL). The chemiluminescence reactions are characterized as having biphasic second order kinetics, CL wavelengths between 350 nm and 650 nm, and exhibiting perturbation by chemicals reactive with singlet oxygen. These data suggest that singlet oxygen species are the excited state responsible for the light emissions. Polarographic studies confirm .O2- consumption and halide release in the reactions, while gas liquid chromatography and NBT reduction demonstrate the decomposition of the halocarbons into products. A chemiluminescent reaction mechanism is proposed involving reductive dehalogenation of the halocarbons and the generation of singlet oxygen. The significance of singlet oxygen generation is discussed with respect to a general mechanism for explaining the rapid initiation of lipid peroxidative membrane damage in halocarbon toxigenicity in animal and plant tissues.

We have compared three competitive chemiluminescent immunoassays (CLIA) for estradiol (E2) using an N-functionalized acridinium ester (AE). The assays were a standard competitive assay using immobilized antibody and directly labeled antigen (type A), an immobilized antibody and indirectly labeled antigen (type B), and an immobilized antigen and labeled antibody (type C). In an antibody-immobilized system, the assay using both AE- and E2-labeled thyroglobulin as a tracer (type B) was more sensitive than that using AE directly coupled with E2 (type A). Subsequently, a comparison of the antibody-immobilized system (type B) and an antigen-immobilized system (type C) showed that the latter was slightly more sensitive than the former. The sensitivity of the CLIA (type C) was similar or superior to commercially available CLIA or radioimmunoassays for E2. Thus, the N-functionalized AE proved to be a useful labeling reagent for a competitive CLIA with high sensitivity.