Principles and applications of luminescence spectrometry coupled to liquid chromatographic techniques

An overview is presented of the physicochemical basis of luminescence, and its application to the detection of chemicals (drugs, biomedically important compounds, environmentally active substances) in liquid chromatographic systems.     

Firefly luciferase: Purification and immobilization

The state of the art of firefly luciferase research is reviewed with special emphasis on its purification and immobilization. The notion of bioluminescence and its role in APT monitoring is described. The need to purify luciferase and the advantages of immobilization are discussed. An insight into the existing methods of luciferase purification and immobilization is given. The scope of the bioluminescent assay is underlined.     

Photochemical Synthesis and Multiphoton Luminescence of Monodisperse Silver Nanocrystals

A rapid, photochemical solution-phase synthesis has been developed for the production of monodisperse, nanometer-sized silver particles. The stabilizer used in the synthesis can be used to control the average diameter of the particles over a range from 1 to 7 nm. The same reaction mixture can also be employed to deposit patterns of nanoparticles with a laser via multiphoton absorption. The particles exhibit strong multiphoton absorption-induced luminescence when irradiated with 800-nm light, allowing emission from single nanoparticles to be observed readily.     

Synthesis, characterization and photophysical properties of mixed ligand tris(polypyridyl)chromium(III) complexes, [Cr(phen)2L]

A series of new heteroleptic, tris(polypyridyl)chromium(III) complexes, [Cr(phen)₂L]³⁺ (L = substituted phenanthrolines or bipyridines), has been prepared and characterized, and their photophyical properties in a number of solvents have been investigated. X-ray crystallography measurements confirmed that the cationic (3+) units contain only one ligand L plus two phenanthroline ligands. Electrochemical and photophysical data showed that both ground state potentials and lifetime decays are sensitive to ligand structure and the nature of the solvent with the exception of compounds containing L = 5-amino-1,10-phenanthroline (aphen) and 2,2′-bipyrimidine (bpm). Addition of electron-donating groups in the ligand structure shifts redox potentials to more negative values than those observed for the parent compound, [Cr(phen)₃]³⁺. Emission decays show a complex dependence with the solvent. The longest lifetime was observed for [Cr(phen)₂(dip)]³⁺ (dip = 4,7-diphenylphenanthroline) in air-free aqueous solutions, τ = 273 μs. Solvent effects are explained in terms of the affinity of hydrophobic complexes for non-polar solvent molecules and the solvent microstructure surrounding chromium units.     

Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.     

Analysis of S-glutathionylated proteins during adipocyte differentiation using eosin-glutathione and glutaredoxin 1

Protein S-glutathionylation is a reversible post-translational modification on cysteine residues forming a mixed disulfide with glutathione. S-glutathionylation, not only protects proteins from oxidation but also regulates the functions of proteins involved in various cellular signaling pathways. In this study, we developed a method for the detection of S-glutathionylated proteins (ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin 1 (mGrx1). ProSSG was efficiently and specifically labeled with E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection of ProSSG with semi-quantitative determination. In addition, a deglutathionylation enzyme mGrx1 specifically released E-GSH from ProSSG-E, which increased fluorescence allowing a sensitive determination of ProSSG levels. Application of the method to the adipocyte differentiation of 3T3-L1 cells showed specific detection of ProSSG and its increase upon differentiation induction, which was consistent with the result obtained by conventional immunoblot analysis, but with greater specificity and sensitivity.     

Osmotic control of luminescence and growth in Photobacterium leiognathi from ponyfish light organs

Osmolarity was found to control the luminescence and growth of Photobacterium leiognathi strain LN-1a isolated from the light organ of the ponyfish Leiognathus nuchalis (family Leiognathidae). Low osmolarity (ca. 400 mOsm) stimulated luminescence per cell 80 to 100-fold to a level (ca. 2.0×10⁴ quanta·s^-1·cell^-1) equal to that of bacteria taken directly from the light organ and increased the level of luciferase per cell 8 to 10-fold compared to high osmolarity (ca. 800 mOsm). Conversely, high osmolarity stimulated oxygen uptake and growth rate 2 to 4-fold compared to low osmolarity. Of 21 additional tested strains of P. leiognathi from light organs of 9 other ponyfish species, all responded similarly. Low osmolarity may be a host control factor that functions to stimulate the luminescence and restrict the growth of ponyfish light organ bacteria in situ.     

Electric-field-induced luminescence emission spectra of Photosystem I and Photosystem II from chloroplasts

The electroluminescence induced by external electric fields in blebs prepared from chloroplasts consists of two kinetically different phases, rapid (R) and slow (S), which were shown to be linked to Photosystem I (PS I) and Photosystem II (PS II) activities, respectively (Symons, M., Korenstein, R. and Malkin, S. (1985) Biochim. Biophys. Acta 806, 305–310). In this report we describe conditions involving heat treatment of broken chloroplasts, which make it possible to observe R phase electroluminescence essentially devoid of any contribution by the S phase. This allowed the precise measurement of the emission spectrum of PS I electroluminescence. The emission spectrum of PS II electroluminescence was obtained using regular broken chloroplasts, which show only S-type emission. The latter emission spectrum is identical to the one obtained for ordinary prompt fluorescence, peaking at 685 nm with a bandwidth of about 25 nm. The PS I emission spectrum is symmetric around 705 nm and is much broader, about 60 nm.     

Synthesis, photophysical and electrochemical properties, and biological labelling studies of luminescent cyclometallated iridium(III) bipyridine-aldehyde complexes

We report the synthesis, characterisation, and photophysical and electrochemical properties of a series of luminescent cyclometallated iridium(III) bipyridine-aldehyde complexes [Ir(N-C)₂(bpy-CHO)](PF₆) (HN-C=2-phenylpyridine, Hppy (1); 2-(4-methylphenyl)pyridine, Hmppy (2); 1-phenylpyrazole, Hppz (3); 3-methyl-1-phenylpyrazole, Hmppz (4); 7,8-benzoquinoline, Hbzq (5); 2-phenylquinoline, Hpq (6); bpy-CHO=4-formyl-4^′-methyl-2,2^′-bipyridine). The X-ray crystal structures of complexes 1 and 4 have been determined. On the basis of the photophysical data, the emission of these complexes is assigned to an excited state of predominantly triplet metal-to-ligand charge-transfer (³MLCT) (dπ(Ir) → (bpy-CHO)) character. For complex 6, the excited state is also mixed with substantial (³IL) () (pq⁻) character. The protein bovine serum albumin has been labelled with these complexes to produce luminescent bioconjugates. The photophysical properties of the luminescent conjugates have also been investigated.