During respiration, it is accepted that oxygen diffuses passively from the lung alveolar spaces through the respiratory epithelium until reaching the pulmonary capillaries, where blood is oxygenated. It is also widely assumed that pulmonary surfactant, a lipid-protein complex secreted into alveolar spaces, has a main surface active function, essential to stabilize the air-liquid interface, reducing in this way the work of breathing. The results of the present work show that capillary water layers containing enough density of pulmonary surfactant membranes transport oxygen much faster than a pure water phase or a water layer saturated with purely lipidic membranes. Membranes reconstituted from whole pulmonary surfactant organic extract, containing all the lipids plus the hydrophobic surfactant proteins, permit also very rapid oxygen diffusion, substantially faster than achieved by membranes prepared from the surfactant lipid fraction depleted of proteins. A model is proposed suggesting that protein-promoted membrane networks formed by pulmonary surfactant might have important properties to facilitate oxygenation through the thin water layer covering the respiratory surface.
The capability of the new luminescent probe (dibenzo[h,j] dipyrido[3,2-a:2',3'-c]phenazine)bis(2,2'-bipyridine)ruthenium(II) dication, (RB2Z), to discriminate live and dead cells has been tested on rat hepatocytes and mouse lymphocytes. RB2Z-stained cells were analyzed using flow cytometry, fluorescence (confocal) microscopy and time-resolved luminescence measurements. The established viability probes propidium iodide (PI) and SYTOX green (SG) were used as controls. The intense luminescence of RB2Z at 606 nm is localized in the nucleus of nonviable cells. Viability measurements by flow cytometry and fluorescence microscopy using RB2Z as dead-cell marker yield the same results as PI and SG. The luminescence lifetime of RB2Z also displays sensitivity to cell viability (0.45 and 0.82 microsecond in presence of fully viable and dead cells, respectively). This ruthenium complex is photostable under laser sources and its 200 nm Stokes shift facilitates multicolor labeling experiments in flow cytometry and fluorescence microscopy. Unlike the currently available probes, the long-lived excited state of RB2Z also allows assays based on luminescence decay measurements.
Homogeneous immunoassays represent an attractive alternative to traditional heterogeneous assays due to their simplicity, sensitivity, and speed. On the basis of a previously identified epitope-mimicking peptide, or mimotope, we developed a homogeneous fluorescence quenching immunoassay based on gold nanoparticles (AuNPs) and a recombinant epitope-mimicking fusion protein for the detection of mycotoxin fumonisin B 1 (FB 1 ). The fumonisin mimotope was cloned as a fusion protein with a yellow fluorescent protein that could be used directly as the tracer for FB 1 detection without the need of labeling or a secondary antibody. Furthermore, owing to the fluorescence quenching ability of AuNPs, a homogeneous immunoassay could be performed in a single step without washing steps to separate the unbound tracer. The homogeneous quenching assay showed negligible matrix effects in 5% wheat extract and high sensitivity for FB 1 detection, with a dynamic range from 7.3 to 22.6 ng mL −1 , a detection limit of 1.1 ng mL −1 , and IC 50 value of 12.9 ng mL −1 , which was significantly lower than the IC 50 value of the previously reported assay using the synthetic counterpart of the same mimotope in a microarray format. The homogeneous assay was demonstrated to be specific for fumonisins B 1 and B 2 , as no significant cross-reactivity with other mycotoxins was observed, and acceptable recoveries (86% for FB 1 2000 μg kg −1 and 103% for FB 1 4000 μg kg −1 ), with relative standard deviation less than 6.5%, were reported from spiked wheat samples, proving that the method could provide a valuable tool for simple analysis of mycotoxin-contaminated food samples.
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