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- Fluorescence sensing of microplastics on surfacesPublication . Costa, Camila Q. V.; Cruz, Joana; Martins, Jorge; Teodosio, Maria; Jockusch, Steffen; Ramamurthy, V.; Da Silva, José PauloMicroplastics, nanoplastics and related products have been recently found in marine ecosystems worldwide, calling for new analytical methods for rapid detection and risk assessment. Fluorescence is a sensitive technique that when used with polarity probes can potentially detect low-polarity plastic particles in environments. Here, we evaluated the fuorescence technique to sense polystyrene microparticles directly on salt, silica and sand surfaces, using Nile Red and pyrene as polarity probes. Results show that all probes displayed fuorescence on silica and sand, whereas strong fuorescence quenching was observed on NaCl. Polystyrene particles increase the fuorescence intensity due to probe migration into their nonpolar microenvironment. In the presence of polystyrene, the spectra of Nile Red are shifted to shorter wavelengths, while the ratio of vibronic bands I1/I3 of fuorescence of pyrene decreases to about 1. Pyrene showed similar sensitivity toward surface-modifed carboxyl polystyrene particles. On NaCl, the emission of pyrene increases linearly with polystyrene content for concentrations from 0.5 to 20 µg/g. The detection limit of polystyrene microparticles on natural sea salt using pyrene as probe is about 0.2 µg/g, while on sand, the sensitivity is about one order of magnitude lower. Overall, although being of relatively low selectivity, the fuorescence technique can be used to determine a maximum content of plastic particles of few micrometers size with little sample preparation. Fluorescence, when used in conjunction with pyrene probe, allows for detection and quantifcation of microplastic particles in the sub-ppm range.
- Analysis of the equilibrium distribution of Ligands in Heterogeneous Media – Approaches and pitfallsPublication . Moreno, Maria João; Loura, Luís M. S.; M. Martins, Jorge; Salvador, Armindo; Velazquez-Campoy, AdrianThe equilibrium distribution of small molecules (ligands) between binding agents in heterogeneous media is an important property that determines their activity. Heterogeneous systems containing proteins and lipid membranes are particularly relevant due to their prevalence in biological systems, and their importance to ligand distribution, which, in turn, is crucial to ligand’s availability and biological activity. In this work, we review several approaches and formalisms for the analysis of the equilibrium distribution of ligands in the presence of proteins, lipid membranes, or both. Special attention is given to common pitfalls in the analysis, with the establishment of the validity limits for the distinct approaches. Due to its widespread use, special attention is given to the characterization of ligand binding through the analysis of Stern–Volmer plots of protein fluorescence quenching. Systems of increasing complexity are considered, from proteins with single to multiple binding sites, from ligands interacting with proteins only to biomembranes containing lipid bilayers and membrane proteins. A new formalism is proposed, in which ligand binding is treated as a partition process, while considering the saturation of protein binding sites. This formalism is particularly useful for the characterization of interaction with membrane proteins.