A generic approach based on long-lifetime fluorophores for the assessment of protein binding to polymer nanoparticles by fluorescence anisotropy.

Quantitation of protein-nanoparticle interactions is essential for the investigation of the protein corona around NPs in vivo and when using synthetic polymer nanoparticles as affinity reagents for selective protein recognition in vitro. Here, a method based on steady-state fluorescence anisotropy measurement is presented as a novel, separation-free tool for the assessment of protein-nanoparticle interactions. For this purpose, a long-lifetime luminescent Ru-complex is used for protein labelling, which exhibits low anisotropy when conjugated to the protein but displays high anisotropy when the proteins are bound to the much larger polymer nanoparticles. As a proof of concept, the interaction of lysozyme with poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid) nanoparticles is studied, and fluorescence anisotropy measurements are used to establish the binding kinetics, binding isotherm and a competitive binding assay.

Ecotoxicity Assessment of Graphene Oxide by Daphnia magna through a Multimarker Approach from the Molecular to the Physiological Level including Behavioral Changes.

The extensive use of engineered nanomaterials, such as graphene oxide (GO), is stimulating research about its potential environmental impacts on the aquatic ecosystem. This study is aimed to comprehensively assess the acute toxicity of a well-characterized GO suspension to Daphnia magna. Conventional ecotoxicological endpoints (lethality, immobilization) and more sensitive, sublethal endpoints (heartbeat rate, feeding activity, and reactive oxygen species (ROS)) production were used. The possible normalization of the heartbeat rate and feeding activity in clean test medium was also investigated. The fate, time-dependent, and concentration-dependent aggregation behaviour of GO was followed by dynamic light scattering, UV-Vis spectroscopy, and zeta potential measurement methods. The EC20 value for immobilization was 50 mg/L, while, for physiological and behavioural endpoints, it ranged from 8.1 mg/L (feeding activity) to 14.8 mg/L (immobilization). The most sensitive endpoint was the ROS production with EC20 = 4.78 mg/L. 24-h recovery experiments revealed that feeding activity was restored only up to a certain level at higher concentrations, indicating that the potential environmental health effects of GO cannot be neglected. Alterations of normal physiology (heart rate) and feeding activity may be associated with increased risk of predation and reproductive decline, highlighting that GO may have impacts on population and food web dynamics in aquatic ecosystems.