Peptide binding domains determined through chemical modification of the side-chain functional groups.

A clear understanding of the specific secondary structure and binding domain resulting from the interactions of proteins and peptides with lipid surfaces will provide insight into the specific functions of biologically active molecules. We have shown in earlier studies that the stationary phases used in reverse-phase high-performance liquid chromatography represent a model artificial lipid surface for the study of induced conformational states of peptides on lipid interaction. We have now used reverse-phase high-performance liquid chromatography to determine the binding domains of peptides and, by extension, of proteins to a lipid surface. This approach consists of performing chemical modifications of specific amino acid side-chain functionalities after the interaction of the peptides with the reverse-phase high-performance liquid chromatography C18 groups. The susceptibility to oxidation was also studied after binding of the same peptides to liposomes. Oxidation of a single methionine residue "walked" through an amphipathic alpha-helical 18-mer peptide was selected to illustrate this approach. The extent of oxidation was found to be clearly dictated by the accessibility of the methionine residue to the aqueous mobile phase. The binding domain found for the peptide in its lipid-induced conformational state was unequivocally the entire hydrophobic face of the amphipathic alpha-helix

Optimization of the Sensitization Process and Stability of Octadentate Eu(III) 1,2-HOPO Complexes

The synthesis of a series of octadentate ligands containing the 1-hydroxypyridin-2-one (1,2-HOPO) group in complex with europium(III) is reported. Within this series, the central bridge connecting two diethylenetriamine units linked to two 1,2-HOPO chromophores at the extremities (5-LIN-1,2-HOPO) is varied from a short ethylene chain (H(2,2)-1,2-HOPO) to a long pentaethylene oxide chain (H(17O5,2)-1,2-HOPO). The thermodynamic stability of the europium complexes has been studied and reveals these complexes may be effective for biological measurements. Extension of the central bridge results in exclusion of the inner-sphere water molecule observed for [Eu(H(2,2)-1,2-HOPO)]- going from a nonacoordinated to an octacoordinated Eu(III) ion. With the longer chain length ligands, the complexes display increased luminescence properties in aqueous medium with an optimum of 20% luminescence quantum yield for the [Eu(H(17O5,2)-1,2-HOPO)]- complex. The luminescence properties for [Eu(H(14O4,2)-1,2-HOPO)]- and [Eu(H(17O5,2)-1,2-HOPO)]- are better than that of the model bis-tetradentate [Eu(5LINMe-1,2-HOPO)2]- complex, suggesting a different geometry around the metal center despite the geometric freedom allowed by the longer central chain in the H(mOn,2) scaffold. These differences are also evidenced by examining the luminescence spectra at room temperature and at 77 K and by calculating the luminescence kinetic parameters of the europium complexes. (Graph Presented)

Time-gated luminescence acquisition for biochemical sensing: miRNA detection*Relacionar en OpeAire*

Luminescence emission is a multidimensional phenomenon comprising a time-domain layer defined by its excited-state kinetics and corresponding lifetime, which is specific to each luminophore and depends on environmental conditions. This feature allows for the discrimination of luminescence signals from species with a similar spectral profile but different lifetimes by time-gating (TG) the acquisition of luminescence. This approach represents an efficient tool for removing unwanted, usually short-lived, signals from scattered light and fluorescence interferents using luminophores with a long lifetime. Due to the emergence of time-resolved techniques using rapid excitation and acquisition methods (i.e., pulsed lasers and single-photon timing acquisition) and new long-lifetime luminophores (i.e., acridones, lanthanide complexes, nanoparticles, etc.), TG analyses can be easily applied to relevant chemical and biochemical issues. The successful application of TG to important biomedical topics has attracted the attention of the R&D industry due to its potential in the development and patenting of new probes, methods and techniques for drug discovery, immunoassays, biomarker discovery and biomolecular interactions, etc. Here, we review the technological efforts of innovative companies in the application of TG-based techniques. Among the many currently available biomarkers, circulating microRNAs (miRNAs) have received attention since they are highly specific and sensitive to different pathological stages of numerous diseases and easily accessible from biological fluids. qPCR is a powerful and routine technique used for the detection and quantification of miRNAs, but qPCR may introduce numerous artefacts and low reproducibility during the amplification process, particularly using complex samples. Thus, due to the efficiency of TG in separating short- lived sources of fluorescence common in biological fluids, TG is an ideal approach for the direct detection of miRNAs in liquid biopsies. Recently, great efforts in the use of TG have been achieved. Our contribution is the proposal of a direct detection approach using TG- imagining with single nucleobase resolution.European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 690866 (miRNA-DisEASY)Proyecto CTQ2017-85658-R. Ministerio de Economía y Competitividad/Agencia Estatal deInvestigación/Fondo Europeo de Desarrollo Regional (FEDER