Nanoparticle-based biosensors are developed and implemented to address a wide range of biomedical questions in bioimaging, molecular diagnostics, targeted drug delivery and controlled release, or photothermal cancer therapy. In this respect, gold nanoparticles provide physicochemical and optical properties that make them particularly interesting. Their relevant, and often unique, plasmonic response has enabled their integration with various transduction modalities (e.g. colorimetric, fluorescence, Raman) and led to the development of extremely sensitive and selective detection platforms. Added advantages associated with gold nanoparticles are their excellent biocompatibility and low toxicity compared to some other nanomaterials (notably quantum dots and silver nanoparticles) when used for sensing and imaging purposes in vivo.In recent years, the performance of gold nanoparticles in the biological milieu has attracted substantial interest. It is well known that, when employing nanoparticles in in vivo studies, proteins present in the biological environment contribute to opsonization, a process that leads to the recognition and attack by macrophages and systematic clearance. For in vitro applications, this process can affect instead the performance of the nanoparticle-based sensors, by reducing in particular sensitivity and selectivity. In both instances, the culprit is the rapid formation of a protein corona around the nanoparticle, which hinders the interaction of any responsive moiety conjugated to its surface with the target molecules present in the surrounding environment. In this talk, I will discuss how the formation of protein corona affects the response of fluorescent flares as they are employed to identify target oligonucleotides in solution. By monitoring the fluorescent response that originates upon hybridization of the nanoflares with their targets, we determined that protein corona, regardless of the identity of the proteins employed to mimic the biological environment, negatively impacts the hybridization dynamics, leading to a reduction in sensitivity by almost 20%, compared to the 2 nM LODs in the absence of corona. We also observed that the presence of protein corona leads to reduced resistance to nucleases, with different effects that depend on the size of the specific proteins, leading us to hypothesize the importance of steric hindrance. Finally, and surprisingly, we observed that polyethylene glycol (PEG) functionalization does not substantially improve the performance of the flares, with further reduced sensitivity due to the lower number of recognition moieties that can be accommodated on the nanoparticle’s surface when PEG is also introduced. We believe that these results will offer further quantitative insight useful for the development of in vivo and in vitro probes based on gold nanoparticles.