Synthesis and characterization of photoactive nanostructured materials for sensing and biomedical applications

1. Promises - In the last decades, nanomaterials have been largely used for sensing and biomedical purposes. The strides that have been made on nanotechnology and the unique physical and chemical properties of nanomaterials offered the possibility to realize tailored devices for almost any analytical purpose. Moreover, nanoscale is the order of magnitude in which biological interactions normally occur. In this way, nanomaterials are specifically realized to image and modify (or both) a biological process. New drug carriers, bioimaging agents and unconventional therapeutics have been adapted to a multitude of biomedical needs. In this scenario, photo-active nanomaterials represent one of the most intriguing products of frontier research. Light possesses high spatio-temporal resolution, photons can be simply produced, controlled, and administered. Among the possible applications, using affordable experimental setups light can be used as an elegant trigger of endergonic processes or to display the fascinating luminescence widely used to probe otherwise difficult-to-see processes. For these reasons, photo-active nanomaterials for chemical sensing and biomedical applications have had a great fortune on the development of diagnostic kits, sensors and nanodrugs. Moreover, and with no less importance, the extensive research on this field allowed to continue reveal the secrets behind the fascinating mechanisms of photophysics that now are even more accessible to the common knowledge in attempt to replicate and understand the more complex, elegant, and efficient processes realized by the nature. 2. Carbon dots - The experimental activity during the PhD period was mainly focused on understanding the behaviour of an emerging class of 0D carbon nanomaterials with interesting photophysical, physical and chemical properties: carbon dots (CDs). 2.1 Carbon dots preparation - Carbon dots were prepared via a bottom-up approach from a natural source recycling the solid waste produced during the olive oil production. The procedure was chosen to be the least impactful to the environment: water was used as solvent, hydrogen peroxide as “green” oxidating agent, hydrothermal procedure was performed under simple reflux for 90 min and carbon precursor is obtained from the valorisation of a food waste. The produced CDs were used for chemical sensing and biomedical purposes. 2.2 Carbon dots conductometric sensor for NOx gas sensing - Interestingly, although the incredibly vast knowledge of chemical sensors based on carbon dots, little was known about their gas sensing capability, in particular as sensing layers onto conductometric devices. It was found high performance sensing activity of carbon dots produced from olive solid wastes coming from the Mediterranean region Puglia (Italy) towards gaseous nitrogen dioxide (NO2). Carbon dots prepared from another Mediterranean region Calabria (Italy) showed instead sensitivity towards gaseous nitrogen monoxide (NO). The different physical and chemical properties of the two CDs types were recognized as mainly dependent from the surface functionalization rate. The surface of carbon dots is abundantly decorated with hydroxyl (-OH), C-O-C, carbonyl (C=O) and carboxylic (-COO-/-COOH) groups that direct the gas sensing capability of carbon dots deposited as sensing layer onto a conductometric device. Spectroscopic and microscopic techniques were used to characterize the two materials and to validate the sensing model already available in literature. CDs produced from Puglia olive solid wastes had a quasi-spherical morphology and an average size of 2.8 ± 0.6 nm (TEM and AFM analysis), their colloidal dispersion in 10 mM PBS showed ζ-potential = -32 mV, thanks to FT-IR spectroscopy -OH, -NH, C-H, C=O, O-C=O, C-O-C groups were recognized and they presented the D and G bands typical of carbon-based materials on Raman spectroscopy. CDs evidenced semiconductor behaviour with a band gap of 1.55 eV determined by cyclic voltammetry experiments. Excellent performances in terms of high and selective response to sub-ppm concentration of NO2 at low temperature (150 °C), low limit of detection (LOD) of 50 ppb, good reproducibility and stability over use and aging were found. CDs produced from Calabria olive solid wastes displayed ζ-potential = -23 mV in water, a complex XRD pattern attributable to graphitic and amorphous nature and a two steps decomposition profile on TGA-DSC analysis were reported. FT-IR, XPS and Raman analyses confirmed the presence of the functional groups previously found on CDs from Puglia olive solid wastes. Comparing the spectroscopic data from the two nanosystems it was speculated that C-O-C and -OH groups are the main functional groups present on CDs from Calabria and Puglia olive solid wastes respectively. The two systems had different optical behaviour on the shifting of the maximum of emission spectra. Carbon dots form Calabria olive solid wastes presented high selective response to NO at 200 °C at 1250 ppm, good stability, and reproducibility. 2.3 The antibacterial activity of carbon dots from olive solid wastes - Inspired by the differences on the physical and chemical properties of carbon dots prepared from olive solid wastes, coming from the Mediterranean regions Puglia and Calabria (Italy), that are mainly dependent from the surface functionalization rate, and in addition with the abundantly demonstrated cytocompatibility in the scientific literature of CDs it was chosen to test their antibacterial activity. It was found overall higher antibacterial activity vs S. Aureus (Gram-positive bacterial strain) than against P. Aeruginosa (Gram-negative bacterial strain). In addition, carbon dots coming from Puglia olive solid waste showed lower activity compared to Calabria CDs. Calabria CDs exhibited bactericidal activity at 360, 240, 120 μg/mL, instead Puglia CDs showed only the 30 % of bacterial cell activity reduction at 360 μg/mL. These results were correlated to the higher surface charge of Calabria CDs compared to Puglia CDs. The two nanosystems had similar size (TEM) and were characterized via UV/Vis, luminescence, FT-IR, and X-ray photoelectron spectroscopies. The optical band gap, using the Tauc plot was calculated. Interestingly, apart the spectral differences, on the physiological conditions, Calabria CDs presented three times more negative ζ-potential than Puglia CDs. 2.4 Carbon dots-Eu(III) as red emitting pH sensor - The variegated surface functional groups of carbon dots, mainly -OH, -COO-, and -NH2 that are responsible of their physical and chemical properties were exploited as complexing moieties for Eu(III) cations. A novel CDs-Eu(III) complex was realized in which carbon dots were utilized as light harvesting antennae inducing an energy transfer onto europium nuclei sensitizing their peculiar, sharp and intense emission in the red region of the electromagnetic spectrum. The sensitization of Eu(III) luminescence, operated by carbon dots, reached the maximum efficiency at the ratio 1:1 (CDs/Eu, w/w). This red emitting complex, useful as potential biosensor because of the lack of interfering optical phenomena such as tissue autofluorescence or non-specific scattering, was tested as a ratiometric luminescent pH sensor (double reading at 456 and 700 nm) on the acidic range. In particular, the luminescence quenching at 700 nm, range in which common fluorescent dyes and indicator does not have characteristic signal, is proportional to pH and attributed to the carboxylic moieties protonation and the consequent Europium displacement. 2.5 Carbon dots-gold nanohybrid - It was continued exploring the excited stated and surface reactivities of CDs realizing a CDs-Au nanohybrid useful for photothermal applications. CDs were functionalized with ethylenediamine to enhance Au(III) complexing and Au(0) binding capabilities. CDs-ethylenediamine excited states, generated by blue light irradiation, served as reducing agent for Au(III) salt to Au(0). In contemporary, CDs-ethylenediamine served as capping agent for the forming gold nanohybrid. The CDs-Au nanohybrid showed comparable photothermal activity to the “gold standard”, the citrate-capped gold nanoparticles firstly prepared by Turkevich. CDs-Au nanohybridization was confirmed via spectroscopic and microscopic techniques. 3. Ag-nanostructured Mg-HA porous scaffold - As complementary activity on noble metals nanomaterials it was also produced a new Mg-hydroxyapatite (Mg-HA) functionalized with silver nanoparticles. The nanofunctionalization was realized in situ, on the surface of the scaffold, via a photochemical reduction of Ag(I) to Ag(0). Results showed exceptional antibacterial activity against E. coli (Gram-negative bacterial strain) and S. Aureus (Gram-positive bacterial strain) but also cytotoxicity on human adipose derived stem cells probably for dysregulated increase of Reactive Oxygen Species (ROS). 4. Silicon nanoparticles from volcanic rocks - During the visiting PhD period at the Laboratory of Nanochemistry of the Institute of Supramolecular Science and Engineering (ISIS) at the University of Strasbourg/CNRS, the scientific activity was focused on the discovery of new nanomaterials starting from unexplored sources. There were produced, by liquid phase exfoliation methods (pH-assisted in water and surfactant-assisted in ethanol), nanostructured materials from light volcanic rocks that are called “lapilli” produced during the explosive activity of Mount Etna (Sicily, Italy). The idea was to apply nanochemistry to volcanic rocks, forge of many elements entrapped in a complex silicate matrix, to produce new nanostructures that may become a new and promising research niche for a plethora of applications in material science, chemistry, and nanomedicine. Heterogeneous types of silicon oxides nanoparticles were produced via liquid phase exfoliation in acidic medium, the more promising of the tested methods. Subsequent size separation was attempted by centrifugation and filtration per steps, monitored via XRD, STEM imaging and spectroscopic methods. According to spectroscopic data (FT-IR, XPS, STEM-EDS) in these nanoparticles are embedded metals such as Al, Fe, Mg, Ca and non-metals such as Cl and C, the latter probably introduced during the exfoliation procedure. Liquid exfoliation treatment produced nanoparticles with an identifiable and characteristic spectral pattern that was different from the non-treated material. Finally, regardless of the size-related heterogeneity, samples according to four-point probe measurements were conductive. They had a work function, estimated by UPS, of approximately 5.3 eV and an optical band gap, estimated by Tauc plot, of ~ 3.2 eV.