Lucio Litti

The development of sustainable microelectronic fabrication strategies is increasingly important due to the environmental and economic limitations of conventional photolithography and vacuum-based metal deposition processes. These approaches typically require hazardous chemicals and high energy consumption. In this work, we address these challenges by introducing an environmental friendly fabrication route for patterning gold microelectrodes, based on inkjet-printed dextran sacrificial masks and spray-coated gold nanoparticles (AuNPs) synthesized via pulsed laser ablation. Inkjet printing enables maskless and material-efficient patterning, while spray coating provides a low-temperature and scalable route for the deposition of conductive nanoparticle networks. The resulting microelectrodes exhibit excellent electrical conductivity (sheet resistance of 37.01 ± 12.09 Ω/square and conductivity of 0.0037 ± 0.0008 MS/cm) and well-defined interdigitated geometries suitable for bioelectronic applications. To evaluate their performance, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are cultured directly on the electrodes. Electrochemical impedance spectroscopy (EIS) is employed as a label-free and real-time technique to monitor cell–electrode interactions and temporal variations in cell coverage and viability. EIS measurements, supported by equivalent circuit modeling, reveal distinct phases corresponding to initial cell adhesion, progressive remodeling of the cellular layer, and long-term reduction in viability, further validated by optical measurements. Overall, this work demonstrates a sustainable and cost-effective fabrication strategy for gold microelectrodes, enabling quantitative, noninvasive monitoring of cellular dynamics through impedance-derived parameters and image-based validation of cell coverage evolution.

The development of theranostic tools for the early detection and localization of tumors represents a major challenge in oncology. Among emerging strategies, the targeting of Programmed Death Ligand-1 (PD-L1), a key immune checkpoint protein overexpressed in many tumor types, has gained significant attention. In this work, we report design and development of theranostic gold nanostructures functionalized with PD-L1-targeting peptides (PTP, sPTP, and rPTP) whose sequences were identified combining structural analysis of the PD-1/PD-L1 interaction interface and molecular dynamics simulations. This is because the design of functional nanostructures for protein targeting requires a precise understanding of how molecular recognition is affected by ligand organization at interfaces; therefore, peptide design was guided not only by the selection of key residues involved in binding but also by the evaluation of peptide assemblies to explicitly account for the collective effects governing target recognition. Indeed, beyond conventional evaluation of protein/single-peptide interaction, peptide clusters and surface-anchored monolayers were investigated to consider features like peptide assembly, organization, and reduced conformational freedom in the nanostructure/PD-L1 interaction. Results indicate that peptide sequence and orientation critically determine monolayer organization and accessibility of the PD-L1 binding motif. The computational predictions were experimentally validated by synthesizing peptide-functionalized gold nanostructures and evaluating their targeting performance against MDA-MB-231 breast cancer cells over-expressing PD-L1, using the surface-enhanced Raman scattering technique: NS functionalized with PTPs achieved the targeting of approximately 85% of MDA-MB-231 cells at 100 pM nanostructure concentration, compared to 23% for those functionalized with rPTP, demonstrating a nearly four-fold difference attributable exclusively to peptide orientation on the nanostructure surface. The specific system investigated in this work establishes a computational framework for the rational design of peptide-functionalized nanostructures, providing insights into the collective behavior of peptide monolayers and offering a smart methodology that, while demonstrated here for targeting PD-L1, is in principle applicable to other protein targets.

Misfolded proteins cause several threatening pathologies, ranging from Alzheimer’s disease to cystic fibrosis. Although several protein folding correctors were tested, their delivery is usually inadequate. Here, via a self-assembly wet reaction, colloidal γ-Fe₂O₃ was used to immobilize two correctors (C4 and C17) for a cystic fibrosis-associated transmembrane protein. The as-obtained core–shell magnetic nanohybrids were extensively characterized, revealing high drug loading and remarkable chemical stability in water. In addition, a dedicated computational study revealed that the whole organic multilayer is involved in a long-range polarization on the nanoconjugate surface, showing sufficient colloidal stability for its application in cells and in contrast with the cargo’s hydrophobic nature. Experiments conducted in HEK293 cells, expressing a mutated subunit of α-sarcoglycan, showed a positive effect on protein complex recovery. This study represents the first in vitro example of a multifunctional nanochaperone for the structural recovery of misfolded proteins.

PD-L1 (programmed cell death ligand-1) is a protein located on the surface of regulatory cells. It has an immunosuppressive role as it binds specifically to the protein programmed cell death-1 (PD-1), a checkpoint glycoprotein, present on the surface of immune cells such as T and B lymphocytes. Many tumor cells block the immune response by overexpressing PD-L1 on their surface; therefore, targeting PD-L1 represents a powerful strategy that allows tumor localization. To determine the presence of PD-L1 in cells, the use of ad hoc functionalized peptides that bind to PD-L1 can be exploited. One of them is the peptide CLP002 (Trp-His-Arg-Ser-Tyr-Tyr-Thr-Trp-Asn-Leu-Asn-Thr), which, bound to surface-enhanced Raman scattering (SERS) gold nanostructures via a suitable linker, was shown to be highly effective in recognizing MDA-MB-231 breast cancer cells and, importantly, this recognition can be measured by SERS experiments. To characterize, on a molecular scale, the interaction between PD-L1 and peptide functionalized nanostructures, we performed molecular dynamics (MDs) simulations, studying the features of peptide monolayers bound on gold surfaces in the absence and presence of PD-L1. The results obtained allow us to explain why the nature of the linker plays a fundamental role in the binding and why a peptide carrying the same amino acids as CPL002 but with a different sequence (scrambled) is much less active than CLP002. These results open the way to an in silico evaluation of the key parameters that regulate the binding of PD-L1 useful for cancer recognition.

This study presents a robust and practical methodology for quantifying the Surface‐Enhanced Raman Spectroscopy (SERS) Enhancement Factor (EF) on both colloidal and solid substrates. SERS, a powerful technique for enhancing the Raman signals of analytes near plasmonic surfaces, has found extensive applications in fields such as biomedicine and materials science. However, inconsistencies in EF measurement methods have hindered cross‐comparisons between different SERS substrates. This work addresses these challenges by providing a standardized protocol for EF determination for a wide variety of substrates composed of gold and silver nanoparticles, as well as nanostructured surfaces. Using p‐mercaptobenzoic acid (p‐MBA) as a model non‐resonant probe, the approach accounts for key variables including substrate geometry, sampling volume, and substrate inhomogeneity. This reliable and reproducible method offers a practical framework for researchers to assess the performance of SERS substrates with simple instruments and techniques that should be easily available in a Raman spectroscopy laboratory.