Gold nanoparticles (AuNPs) show physicochemical and optical functionalities that are of great interest for spectroscopy-based detection techniques, and especially for surface enhanced Raman spectroscopy (SERS), which is capable of providing detailed information on the molecular content of analysed samples. Moreover, the introduction of different moieties combines the interesting plasmonic properties of the AuNPs with the specific and selective recognition capabilities of the antibodies (Ab) towards antigens. The conjugation of biomolecules to gold nanoparticles (AuNPs) has received considerable attention for analysis of liquid samples and in particular biological fluids (biofluids) in clinical diagnostic and therapeutic field. To date, gold nanostars (AuNSts) are gaining more and more attention as optimal enhancers for SERS signals due to the presence of sharp branches protruding from the core, providing a huge number of “hot spots”. To this end, we focused our attention on the design, optimization, and deep characterization of a bottom up-process for (i) AuNPs increasing stabilization in high ionic strength buffer, (ii) covalent conjugation with antibodies, while (iii) retaining the biofunctionality to specific tag analyte within the biofluids. In this work, a SERS-based substrate was developed for the recognition of a short fragment (HA) of the hemagglutinin protein, which is the major viral antigen inducing a neutralizing antibody response. The activity and specific targeting with high selectivity of the Ab-AuNPs was successfully tested in transfected neuroblastoma cells cultures. Then, SERS capabilities were assessed measuring Raman spectra of HA solution, thus opening interesting perspective for the development of novel versatile highly sensitive biofluids sensors.

Trodusquemine is an aminosterol known to prevent the binding of misfolded protein oligomers to cell membranes and to reduce their toxicity in a wide range of neurodegenerative diseases. Its precise mechanism of action, however, remains unclear. To investigate this mechanism, we performed confocal microscopy, fluorescence resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) measurements, which revealed a strong binding of trodusquemine to large unilamellar vesicles (LUVs) and neuroblastoma cell membranes. Then, by combining quartz crystal microbalance (QCM), fluorescence quenching and anisotropy, and molecular dynamics (MD) simulations, we found that trodusquemine localises within, and penetrates, the polar region of lipid bilayer. This binding behaviour causes a decrease of the negative charge of the bilayer, as observed through ζ potential measurements, an increment in the mechanical resistance of the bilayer, as revealed by measurements of the breakthrough force applied with AFM and ζ potential measurements at high temperature, and a rearrangement of the spatial distances between ganglioside and cholesterol molecules in the LUVs, as determined by FRET measurements. These physicochemical changes are all known to impair the interaction of misfolded oligomers with cell membranes, protecting them from their toxicity. Taken together, our results illustrate how the incorporation in cell membranes of sterol molecules modified by the addition of polyamine tails leads to the modulation of physicochemical properties of the cell membranes themselves, making them more resistant to protein aggregates associated with neurodegeneration. More generally, they suggest that therapeutic strategies can be developed to reinforce cell membranes against protein misfolded assemblies.

We have studied two misfolded oligomeric forms of the protein HypF-N, which show similar morphologies but very different toxicities.

Background: Early diagnosis is essential in the field of lysosomal storage disorders for the proper management of patients and for starting therapies before irreversible damage occurs, particularly in neurodegenerative conditions. Currently, specific biomarkers for the diagnosis of lysosomal storage disorders are lacking in routine laboratory practice, except for enzymatic tests, which are available only in specialized metabolic centers. Recently, we established a method for measuring and verifying changes in GM1 ganglioside levels in peripheral blood lymphocytes in patients with GM1 gangliosidosis. However, fresh blood is not always available, and using frozen/thawed lymphocytes can lead to inaccurate results. Methods: We used frozen/thawed fibroblasts obtained from stored biopsies to explore the feasibility of fluorescent imaging and flow-cytometric methods to track changes in storage materials in fibroblasts from patients with three lysosomal neurodegenerative conditions: GM1 gangliosidosis, Sialidosis, and Niemann–Pick type C. We used specific markers for each pathology. Results and Conclusions: We demonstrated that with our methods, it is possible to clearly distinguish the levels of accumulated metabolites in fibroblasts from affected and unaffected patients for all the three pathologies considered. Our methods proved to be rapid, sensitive, unbiased, and potentially applicable to other LSDs.

Multifunctional magneto-plasmonic nanoparticles (MP-NPs) are attracting increasing interest for biomedical applications due to their dual magnetic and optical properties. However, existing synthesis protocols for MP-NPs could be limited by harsh conditions or lengthy, complex procedures. These limitations can hinder the development of nanosystems that work effectively in biological dispersion. In this work, we present a flexible nanoplatform for capture and probe applications, based on MP-NPs and bioorthogonal plasmonic nanoparticles (AuNPs). This system enables efficient cell sorting and surface-enhanced Raman scattering (SERS) detection in complex biological media under dynamic conditions. Magnetic Fe₃O₄ nanoclusters were synthesized using a scalable, aqueous Massart-based protocol. These structures were then densely decorated with electrostatically assembled AuNPs, producing magnetically responsive and plasmonically active nanocomposites with high aqueous stability. A polyethylene glycol (PEG) derivative was used for surface functionalization, improving colloidal stability and enabling broad bio-recognition capability. To enhance detection sensitivity and shift the optical response into the near-infrared (NIR) range, bioorthogonal Raman-active gold nanostars (AuNSts) were integrated into a sandwich configuration with the MP-NPs. The system demonstrated selective sorting and sensitive SERS-based identification of cell subpopulation within a heterogeneous sample. Additionally, the integration of the MP-NPs assay into a 3D-printed microfluidic device allowed controlled nanoparticles manipulation using external magnets, enhancing magnetic separation, hot-spots generation, and assay reusability. Overall, these MP-NPs platform combines scalable synthesis, tunable colloidal and surface properties, and compatibility with microfluidic systems. This work lays the foundation for advanced biosensing approaches in lab-on-chip formats for clinical diagnostics and targeted analysis of rare cell populations.