An HPLC method for the determination of selected amino acids in human embryo culture medium

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra‐cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at −80°C. Filtered medium samples were derivatized with ortho ‐phthalaldehyde (naphthalene‐2,3‐dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse‐phase columns (LichroCART, Purospher STAR RP_18e or Ascentis Express C₁₈) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra‐assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.     

Sensitive and selective detection of alcohols via fluorescence modulation

Reported herein is the selective detection of aliphatic alcohols using cyclodextrin-promoted, proximity-induced fluorescence modulation of a high-quantum yield fluorophore. This fluorescence modulation occurred when the analyte was held in close proximity to the fluorophore via non-covalent cyclodextrin–analyte–fluorophore interactions, and led to unique modulation responses for each analyte, fluorophore and cyclodextrin investigated. These changes in fluorescence were used for the generation of an array using linear discriminant analysis that successfully generated unique pattern identifiers for 99% of the investigated analytes, and could detect alcohols at micromolar concentrations. These results represent a fundamentally new detection approach for these challenging analytes, and have significant potential in the development of novel detection schemes.     

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

Despite the fact that transmembrane proteins represent the main therapeutic targets for decades, complete and in-depth knowledge about their biochemical and pharmacological profiling is not fully available. In this regard, target-tailored small-molecule fluorescent ligands are a viable approach to fill in the missing pieces of the puzzle. Such tools, coupled with the ability of high-precision optical techniques to image with an unprecedented resolution at a single-molecule level, helped unraveling many of the conundrums related to plasma proteins’ life-cycle and druggability. Herein, we review the recent progress made during the last two decades in fluorescent ligand design and potential applications in fluorescence microscopy of voltage-gated ion channels, ligand-gated ion channels and G-coupled protein receptors.     

Polydiacetylene‐Tb3+ Nanosheets of Which Both the Color and the Fluorescence Can Be Reversibly Switched between Two Colors

Polydiacetylenes (PDAs), an organic layered compound, show a series of intriguing properties, such as thermochromism and fluorescence emission in the red‐phase. However, their irreversible color change, and weak and single‐color fluorescence emitted only from the red‐phase PDAs, have limited their applications. Herein, we report double‐reversible PDA‐Tb³⁺ nanosheets of which both the color and the fluorescence can be reversibly switched between two colors. PDA‐Tb³⁺ nanosheets have the nearly defect‐free intercalated structure in which a layer of Tb³⁺ ions was intercalated in between each two PDA bilayers to tether almost all of the carboxyl groups at the end of the side chains of the PDA. When the PDA is in the blue phase, the PDA‐Tb³⁺ nanosheets emit the green fluorescence of Tb³⁺ ions. When the PDA is in the red phase, the Tb³⁺ fluorescence disappears while the intrinsic red fluorescence of PDA is effectively enhanced through the fluorescence resonance energy transfer (FRET) process; the PDA‐Tb³⁺ nanosheets emit stronger red fluorescence compared with the PDA in red phase. Moreover, the tethering of almost all of the carboxyl groups at the end of the side chains of the PDA endows the nanosheets with the double reversibility in both the color and fluorescence transitions.     

In situ studies of microbial inactivation during high pressure processing

High pressure processing (HPP) has been shown to reduce microbial concentration in foods. The mechanisms of microbial inactivation by HPP have been associated with damage to cell membranes. The real-time response of bacteria to HPP was measured to elucidate the mechanisms of inactivation, which can aid in designing more effective processes. Different pressure cycling conditions were used to expose Enterobacter aerogenes cells to HPP. Propidium iodide (PI) was used as a probe, which fluoresces after penetrating cells with damaged membranes and binding with nucleic acids. A HPP vessel with sapphire windows was used for measuring fluorescence in situ. Membrane damage was detected during pressurization and hold time, but not during depressurization. The drop in fluorescence was larger than expected after pressure cycles at higher pressure and longer times. This indicated possible reversible disassociation of ribosomes resulting in additional binding of PI to exposed RNA under pressure and its release after depressurization.     

One‐pot direct synthesis of fluorescent polyaniline‐porphyrin macrospheres from porphyrin

Porphyrins are very important chromophores as sensitizer in solar cell. Hole and electron transport layers are being used as an important layer to move the electrons and holes away from the sensitizer molecule to electrodes. In this work, a simple process has been developed for the synthesis of one layer combination of sensitizer and hole transport, that is, polyaniline‐porphyrin (PANI‐TPPS₄) system as a bulk heterojunction. Macrospheres of fluorescent PANI‐TPPS₄ materials were synthesized by one‐pot as well as two step processes directly from porphyrin for the first time. The advantage of this process is no need to isolate sulfonated porphyrin (TPPS₄), which is a difficult process. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Validity of the quantum regression theorem for resonance fluorescence in a photonic crystal

Correlation functions of a driven two‐level system embedded in a photonic crystal are analyzed. The spectral density of the photonic bands near a gap makes this system non‐Markovian. The equations of motion for two‐time correlations are derived by two different methods, the quantum regression theorem and the fluctuation dissipation theorem, and found to be the same.     

LR12‐peptide quantitation in whole blood by RP‐HPLC and intrinsic fluorescence detection: Validation and pharmacokinetic study

A simple, sensitive, selective and robust HPLC method based on intrinsic fluorescence detection was developed for the quantitation of a dodecapeptide (designated as LR12), inhibitor of Triggering Receptor Expressed on Myeloid cells‐1, in rat whole blood. Sample treatment was optimized using protein precipitation and solid‐phase extraction. Chromatographic separation was carried out in a gradient mode using a core–shell C₁₈ column (150 × 4.6 mm, 3.6 μm) with mobile phases of acetonitrile and water containing trifluoroacetic acid at 1.0 mL/min. The method was validated using methodology described by the US Food and Drug Administration guidelines for bioanalytical methods. Linearity was demonstrated within the 50–500 ng/mL range and the lower limit of quantitation was 50 ng/mL. Finally, a preliminary pharmacokinetic study after intraperitoneal injection of LR12 in rats was conducted to evaluate both LR12 monomer and its corresponding disulfide dimer, the main product of degradation. Beyond the fact that this paper describes the first fully validated method for LR12 analysis in blood samples, the approach followed here to optimize pre‐analytical steps could be beneficial to develop HPLC and/or MS methods for other pharmaceutical peptides.