Photo-responsive antibacterial surfaces combining both on-demand photo-switchable activity and sustained biocidal release were prepared using sequential chemical grafting of nano-objects with different geometries and functions. The multi-layered coating developed incorporates a monolayer of near-infrared active silica-coated gold nanostars (GNS) decorated by silver nanoparticles (AgNP). This modular approach also enables us to unravel static and photo-activated contributions to the overall antibacterial performance of the surfaces, demonstrating a remarkable synergy between these two mechanisms. Complementary microbiological and imaging evaluations on both planktonic and surfaceattached bacteria provided new insights on these distinct but cooperative effects.The development of smart on-demand antimicrobial surfaces has become a critical research area, addressing the issue of indiscriminate use of biocides implicated in the global emergence of antimicrobial resistance 1 . A significant challenge in this field is the need to combine on-demand antimicrobial functions at surfaces with static long-lasting effects. Static antibacterial surfaces are generally based on the release of metal cations with intrinsic biocidal properties 2 , e.g. sustained Ag + release from surface-attached silver nanoparticles [3][4][5] . In this area we have recently demonstrated that after 24 h such surfaces can reduce the colony forming units (CFU) of planktonic Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) by 5-7 orders of magnitude 4 and reduce the surviving fraction of Staphylococcus epidermidis (S. epidermidis) biofilms by 5 orders of magnitude 5 . Alternatively, the use of light-activated antimicrobial surfaces provides a route towards responsive systems that can be triggered remotely and on-demand 6 . Translation of such technology to in vivo applications (e.g. on the surfaces of prostheses and implants) needs to be restricted to systems responding in the narrow near-infrared (NIR) spectral window (750-900 nm) in which water and living tissues are transparent and, therefore, not susceptible to damage [7][8][9][10][11] . In this context, photothermal conversion by non-spherical plasmonic nanoparticles (e.g. nanorods 12 or nanostars) 7 is becoming particularly interesting, allowing the use of local hyperthermia activated by NIR radiation to kill bacteria. In 2014, the first example of combined use of biocidal metal cations and photothermal conversion of light was published 13 , using biomimetically-coated Au/Ag core/shell nanorods, and demonstrating a synergistic antibacterial effect against planktonic E. coli and S. epidermidis bacteria upon NIR irradiation. More recently, we have developed a different approach, specially designed for surfaces, where glass substrates bearing monolayers of non-coated Ag nanoplates 14 or Ag nanotriangles 15 displayed antibacterial effects significantly enhanced upon NIR irradiation with respect to the intrinsic biocidal effects due to the release of Ag + cations. Remarkably, the amou...

Surface modification of noble metal nanoparticles with mixed molecular monolayers is one of the most powerful tools in nanotechnology, and is used to impart and tune new complex surface properties. In imaging techniques based on surface enhanced Raman spectroscopy (SERS), precise and controllable surface modifications are needed to carefully design reproducible, robust and adjustable SERS nanoprobes. We report here the attainment of SERS labels based on gold nanostars (GNSs) coated with a mixed monolayer composed of a poly ethylene glycol (PEG) thiol (neutral or negatively charged) that ensure stability in biological environments, and of a signalling unit 7-Mercapto-4-methylcoumarin as a Raman reporter molecule. The composition of the coating mixture is precisely controlled using an original method, allowing the modulation of the SERS intensity and ensuring overall nanoprobe stability. The further addition of a positively charged layer of poly (allylamine hydrocloride) on the surface of negatively charged SERS labels does not change the SERS response, but it promotes the penetration of GNSs in SH-SY5Y neuroblastoma cells. As an example of an application of such an approach, we demonstrate here the internalization of these new labels by means of visualization of cell morphology obtained with SERS mapping.

The adhesion and proliferation of bacteria on abiotic surfaces pose challenges in both health care and industrial applications. Gold nanostars (GNSs) monolayers grafted on glass have demonstrated to exert antibacterial action due to their photo-thermal features. Here, these GNS layers were further functionalized using thiols monolayers, in order to impart different wettability to the surfaces and thus adding a feature that could help to fight bacterial proliferation. Thiol that has different functional groups was used and the thiol-protected surfaces were characterized by means of UV-vis spectroscopy, contact angles, SEM and surface enhanced Raman spectroscopy (SERS). We verified that (i) coating with the proper thiol allows us to impart high hydrophilicity or hydrophobicity to the surfaces (with contact angle values ranging from 10 to 120°); (ii) GNS monolayers are strongly stabilized by functionalization with thiols, with shelf stability increasing from a few weeks to more than three months and (iii) photo-thermal features and subsequent antibacterial effects caused by hyperthermia are not changed by thiols layers, allowing us to kill at least 99.99% of representative bacterial strains.

We prepared and characterized recyclable surface enhanced Raman spectroscopy (SERS) active glass chips. Gold nanostars were grafted on properly functionalized glasses by means of electrostatic interactions and then they were coated with a silica layer of controllable thickness in the nanometer range. The SERS activity of the obtained substrates were tested in terms of reproducibility and homogeneity intra-samples and inter-samples from different batches using the Raman reporter as the model compound rhodamine 6G. The uncoated substrates were used as reference to evaluate the effect of silica spacers on SERS enhancement factors (EFs). The chemical route to obtain silica-coated SERS chips is described in detail, and the morphology and the optical response of substrates have been characterized. We demonstrate that SERS substrates coated with 1 nm silica conserve a good EF, and that the coating confers to the SERS platform an extreme robustness leading to reusability of the substrates.

The last decade has come across an increasing demand for theranostic biocompatible nanodevices possessing the double ability of diagnosis and therapy. In this work, we report the design, synthesis and step-by-step characterization of rationally coated gold nanostars (GNSs) for the SERS imaging and photothermal therapy of HeLa cancer cells. The nanodevices were realized by synthesizing GNSs with a seed growth approach, coating them with a controlled mixture of thiols composed of a Raman reporter and a polyethylene glycol with a terminal amino group, and then reacting these amino groups with folic acid (FA), in order to impart selectivity towards cancer cells which overexpress folate receptors on their membranes. After a complete characterization, we demonstrate that these FA-functionalized GNSs (FA-GNSs) are able to bind selectively to the membranes of HeLa cells, acting as SERS tags and allowing SERS imaging. Moreover, we demonstrate that once bound to HeLa cell membranes, FA-GNSs exhibit photothermal effect which can be exploited to kill the same cells in vitro using laser irradiation in the NIR at a very low and safe irradiance. We thus demonstrate that the FA-GNSs designed following the described approach are an efficient prototype of theranostic nanodevices.